Alexander's legacy includes the cultural diffusion and syncretism which his conquests engendered, such as Greco-Buddhism. He founded some twenty cities that bore his name, most notably Alexandria in Egypt. Alexander's settlement of Greek colonists and the resulting spread of Greek culture in the east resulted in a new Hellenistic civilization, aspects of which were still evident in the traditions of the Byzantine Empire in the mid-15th century AD and the presence of Greek speakers in central and far eastern Anatolia until the 1920s. Alexander became legendary as a classical hero in the mold of Achilles, and he features prominently in the history and mythic traditions of both Greek and non-Greek cultures. He became the measure against which military Leaders compared themselves, and military academies throughout the world still teach his tactics. He is often ranked among the most influential people in history.

Alexander's legacy extended beyond his military conquests. His campaigns greatly increased contacts and trade between East and West, and vast areas to the east were significantly exposed to Greek civilization and influence. Some of the cities he founded became major cultural centers, many surviving into the 21st century. His chroniclers recorded valuable information about the areas through which he marched, while the Greeks themselves got a sense of belonging to a world beyond the Mediterranean.

After the death of Spitamenes and his marriage to Roxana (Raoxshna in Old Iranian) to cement relations with his new satrapies, Alexander turned to the Indian subcontinent. He invited the chieftains of the former satrapy of Gandhara (a region presently straddling eastern Afghanistan and northern Pakistan), to come to him and submit to his authority. Omphis (Indian name Ambhi), the ruler of Taxila, whose kingdom extended from the Indus to the Hydaspes (Jhelum), complied, but the chieftains of some hill clans, including the Aspasioi and Assakenoi sections of the Kambojas (known in Indian texts also as Ashvayanas and Ashvakayanas), refused to submit. Ambhi hastened to relieve Alexander of his apprehension and met him with valuable presents, placing himself and all his forces at his disposal. Alexander not only returned Ambhi his title and the gifts but he also presented him with a wardrobe of "Persian robes, gold and silver ornaments, 30 horses and 1,000 talents in gold". Alexander was emboldened to divide his forces, and Ambhi assisted Hephaestion and Perdiccas in constructing a bridge over the Indus where it bends at Hund (Fox 1973), supplied their troops with provisions, and received Alexander himself, and his whole army, in his capital city of Taxila, with every demonstration of friendship and the most liberal hospitality.

Several natural causes (diseases) have been suggested, including malaria and typhoid fever. A 1998 article in the New England Journal of Medicine attributed his death to typhoid fever complicated by bowel perforation and ascending paralysis. Another recent analysis suggested pyogenic (infectious) spondylitis or meningitis. Other illnesses fit the symptoms, including acute pancreatitis and West Nile virus. Natural-cause theories also tend to emphasize that Alexander's health may have been in general decline after years of heavy drinking and severe wounds. The anguish that Alexander felt after Hephaestion's death may also have contributed to his declining health.

The strongest argument against the poison theory is the fact that twelve days passed between the start of his illness and his death; such long-acting poisons were probably not available. However, in a 2003 BBC documentary investigating the death of Alexander, Leo Schep from the New Zealand National Poisons Centre proposed that the plant white hellebore (Veratrum album), which was known in antiquity, may have been used to poison Alexander. In a 2014 manuscript in the journal Clinical Toxicology, Schep suggested Alexander's wine was spiked with Veratrum album, and that this would produce poisoning symptoms that match the course of events described in the Alexander Romance. Veratrum album poisoning can have a prolonged course and it was suggested that if Alexander was poisoned, Veratrum album offers the most plausible cause. Another poisoning explanation put forward in 2010 proposed that the circumstances of his death were compatible with poisoning by water of the river Styx (modern-day Mavroneri in Arcadia, Greece) that contained calicheamicin, a dangerous compound produced by bacteria.

Greek Historian Arrian (Lucius Flavius Arrianus 'Xenophon' c. 86 – c. 160 AD) described Alexander as:

Some of the most pronounced effects of Hellenization can be seen in Afghanistan and India, in the region of the relatively late-rising Greco-Bactrian Kingdom (250–125 BC) (in modern Afghanistan, Pakistan, and Tajikistan) and the Indo-Greek Kingdom (180 BC – 10 AD) in modern Afghanistan and India. There on the newly formed Silk Road Greek culture apparently hybridized with Indian, and especially Buddhist culture. The resulting syncretism known as Greco-Buddhism heavily influenced the development of Buddhism and created a culture of Greco-Buddhist art. These Greco-Buddhist kingdoms sent some of the first Buddhist missionaries to China, Sri Lanka, and the Mediterranean (Greco-Buddhist monasticism). Some of the first and most influential figurative portrayals of the Buddha appeared at this time, perhaps modeled on Greek statues of Apollo in the Greco-Buddhist style. Several Buddhist traditions may have been influenced by the ancient Greek religion: the concept of Boddhisatvas is reminiscent of Greek Divine heroes, and some Mahayana ceremonial practices (burning incense, gifts of flowers, and food placed on altars) are similar to those practiced by the ancient Greeks; however, similar practices were also observed amongst the native Indic culture. One Greek king, Menander I, probably became Buddhist, and was immortalized in Buddhist literature as 'Milinda'. The process of Hellenization also spurred trade between the east and west. For Example, Greek astronomical instruments dating to the 3rd century BC were found in the Greco-Bactrian city of Ai Khanoum in modern-day Afghanistan, while the Greek concept of a spherical earth surrounded by the spheres of planets eventually supplanted the long-standing Indian cosmological belief of a disc consisting of four continents grouped around a central mountain (Mount Meru) like the petals of a flower. The Yavanajataka (lit. Greek astronomical treatise) and Paulisa Siddhanta texts depict the influence of Greek astronomical ideas on Indian astronomy.

Alexander was erudite and patronized both arts and sciences. However, he had little interest in Sports or the Olympic games (unlike his father), seeking only the Homeric ideals of honour (timê) and glory (kudos). He had great charisma and force of personality, characteristics which made him a great leader. His unique abilities were further demonstrated by the inability of any of his generals to unite Macedonia and retain the Empire after his death—only Alexander had the ability to do so.

The core of the Hellenistic culture promulgated by the conquests was essentially Athenian. The close association of men from across Greece in Alexander's army directly led to the emergence of the largely Attic-based "koine", or "common" Greek dialect. Koine spread throughout the Hellenistic world, becoming the lingua franca of Hellenistic lands and eventually the ancestor of modern Greek. Furthermore, town planning, education, local government, and art current in the Hellenistic period were all based on Classical Greek ideals, evolving into distinct new forms commonly grouped as Hellenistic. Aspects of Hellenistic culture were still evident in the traditions of the Byzantine Empire in the mid-15th century.