Hema was a supporting artiste in Idhu Sathiyam (1963) and portrayed a danseuse in Pandava Vanavasam (1965). In 1968, Malini played opposite Raj Kapoor in Sapno Ka Saudagar. That was the beginning of her promotion as the "Bollywood dream girl."

In Johnny Mera Naam (1970), Malini took a lead role and she took other challenging roles, for Example, a young widow in Andaz (1971) and a jealous woman in a negative character in Lal Patthar (1971). In 1972, Malini played opposite Dharmendra and Sanjeev Kumar in Seeta Aur Geeta. Malini received a Filmfare Best Actress Award for this film. Within four years of making her début, Malini was an established lead Actress in Bollywood and classical Dancer. Malini's films of the 1970s include Sanyasi (1975), Dharmatma and Pratigya (1975). In Sholay (1975), Malini played the role of Basanti, a talkative girl. Other works of the period include Trishul, Joshila, Khushboo (1975), Kinara (1977) and Meera (1979).

During her career, she received eleven nominations for the Filmfare Award for Best Actress, winning the award in 1973. In 2000, Malini won the Filmfare Lifetime Achievement Award and also the Padma Shri, the fourth-highest civilian honour awarded by the Government of India. In 2012, the Sir Padampat Singhania University conferred an Honorary Doctorate on Malini in recognition of her contribution to Indian cinema. Malini served as chairperson of the National Film Development Corporation. In 2006, Malini received the Sopori Academy of Music And Performing Arts (SaMaPa) Vitasta award from Bhajan Sopori in Delhi for her contribution and Service to Indian culture and dance. In 2013, she received the NTR National Award from the Government of Andhra Pradesh for her contribution to Indian cinema.

Her screen presence and chemistry with Rajesh Khanna was appreciated in the film Prem Nagar (1974 film). Malini portrayed the role of a classical singer in the critically acclaimed film Mehbooba opposite Rajesh Khanna, and the Times of India called them one of the top film couples. The subsequent films of the pair; Palkon Ki Chhaon Mein and Janta Hawaldar became box office flops on their release, though over the years these films have achieved cult status.

Her commercially successful films, after 1980, with Rajesh Khanna included Dard, Bandish, Kudrat, Hum Dono, Rajput, Babu, Durgaa, Sitapur Ki Geeta and Paap Ka Ant.

After her marriage, Malini made films including Kranti, Naseeb (1981), Satte Pe Satta, Rajput, and Ek Nai Paheli (1984). Her works also included Aandhi Toofan, Durgaa (1985), Ramkali (1985), Sitapur Ki Geeta (1987), Ek Chadar Maili Si (1986), Rihaee and Jamai Raja (1990).

During this period, Malini performed with Dharmendra in Alibaba Aur 40 Chor, Baghavat, Samraat, Razia Sultan, Andha Kanoon (1983), Baghavat, and Raaj Tilak (1984).

In the 1990s, she produced and directed the 1992 film Dil Aashna Hai, starring Divya Bharti and Shahrukh Khan in the leading roles. She also produced and directed her second feature film Mohini (1995), starring her niece Madhoo and actor Sudesh Berry in the leading roles. She then focused on dancing and television work, only occasionally appearing in films. In 1997, she acted in Vinod Khanna's production Himalay Putra which marked the debut of Vinod's son Akshaye Khanna.

Malini's niece is Actress Madhoo Raghunath, who played the female lead in Phool Aur Kaante (1991), Roja (1992) and Annayya (1993).

In 1999, Malini campaigned for the Bharatiya Janata Party (BJP) candidate, Vinod Khanna, a former Bollywood actor, in the Lok Sabha Elections in Gurdaspur, Punjab. In February 2004, Malini officially joined the BJP. From 2003 to 2009, she served as an MP to the upper house – the Rajya Sabha, having been nominated by the then President of India, Dr. A.P.J. Abdul Kalam. In March 2010, Malini was made general secretary of the BJP, and in February 2011, she was recommended by Ananth Kumar, the party general secretary. In the 2014 general elections for the Lok Sabha, Malini defeated the Mathura incumbent, Jayant Chaudhary (RLD) by 3,30,743 votes. Malini was then elected to the Lok Sabha.

Malini was the Editor of New Woman and Meri Saheli, Hindi women's magazine. In 2000, Malini was appointed as the first female chairperson of the National Film Development Corporation for a term of three years.

From 2003 to 2009, Malini was elected to the Rajya Sabha, the upper House of parliament, as a representative of the Bharatiya Janata Party. In 2014, Malini was elected to the Lok Sabha. Malini has been involved with charitable and social ventures. Currently, Malini is also a life member of the International Society for Krishna Consciousness (ISKCON). She was the top successful Actress of her time. She has been praised by critics for her dancing and acting.

In 2007, Malini entered a promotional contract with Kent RO Systems, makers of a mineral water purifier system. Malini also became a brand ambassador for Pothys, a textile showroom in Chennai.

Malini is a supporter of the animal rights organisation, PETA India. In 2009, she wrote a letter to the Mumbai Municipal Commissioner urging him to ban horse carriages from Mumbai`s busy streets. In 2011, she wrote to the Union Minister for Environment and Forests, Jairam Ramesh, urging him to ban bull fighting (jallikattu). She said, "My friends at PETA have organised investigations at jallikattu events and documented that bulls are pulled roughly by their nose rings, punched, pummelled, hit with sharp sticks and crammed into trucks so tightly that they can barely move". Malini won the title of "PETA Person of the Year". As a vegetarian, she said, "Knowing that my food choices are helping the planet and animals too, makes me happy".

Malini was the third child born of a Tamil-speaking Iyengar family to her mother Jaya Lakshmi Chakravarti, a film Producer, and VSR Chakravarti. Malini attended the Andhra Mahila Sabha in Chennai where her favourite subject was history. Malini studied at DTEA Mandir Marg (Doubtful), and in 11th Standard she left to begin her acting career.

On 22 April 2017, Malini said she would take action against Maharashtra independent MLA Omprakash Babarao Kadu for making derogatory comments against her earlier days.