After taking ten years to graduate from the Hochschule für Welthandel (now Vienna University of Economics and Business Administration) with a marketing degree, Mateschitz's first employer was Unilever, where he worked marketing detergents. He subsequently moved to Blendax, the German cosmetics company since bought by Procter & Gamble, where he worked on, among other things, the marketing of Blendax toothpaste. It was as part of his travels for Blendax that he discovered Krating Daeng, the drink that would later become Red Bull. In 1984, he founded Red Bull GmbH with his Thai partners Chaleo and his eldest son Chalerm Yoovidhya, with the launch in Austria in 1987. Subsequently, he turned the Red Bull drink into a world market leader among Energy drinks.

Mateschitz's brands are consistently marketed as associated with the physical and mental attributes needed for various types of extreme Sports through commercial sponsorship. Red Bull formerly owned more than 60 percent of the Sauber Formula One motor racing team, and was the team's main sponsor. However, Red Bull ended its relationship with Sauber at the end of 2001 after the team opted to sign Kimi Räikkönen as one of their drivers for the 2001 season instead of Red Bull protege Enrique Bernoldi. In November 2004, Mateschitz bought the Jaguar Racing Formula One team from its previous owners, Ford, and renamed it Red Bull Racing. In September 2005, Mateschitz joined forces with his close friend and former Formula One driver, Gerhard Berger, to purchase the Italian-registered Minardi team from its Australian owner Paul Stoddart. The team was renamed Scuderia Toro Rosso, Toro Rosso meaning Red Bull in Italian. In 2010, Red Bull Racing won the Formula One World Constructors' Championship and Drivers' Championship with Sebastian Vettel. They then went on to win both titles for the next three years running in 2011, 2012, and 2013, making Vettel and Red Bull Racing four-time world champions.

In April 2005, he bought the Austrian football club SV Austria Salzburg and in March 2006, he bought the American club MetroStars; both clubs were subsequently renamed after his famous drink, as Red Bull Salzburg and New York Red Bulls, respectively. In 2007, Red Bull founded Red Bull Brasil, a football team based in Campinas, Brazil. The team was promoted to the 1st division of the São Paulo championship in 2014, the most competitive state championship of the country. In May 2009, he founded a German football club called RB Leipzig and was named as owner. Since 2012, he is also the owner of the German ice hockey club EHC München, which also changed its name into Red Bull München.

From 2006 to 2011, Mateschitz also owned Team Red Bull who competed in the NASCAR Sprint Cup Series and the K&N Pro Series East.

He lives in Fuschl am See, Austria but also owns Laucala Island, off Fiji, which he bought from the Forbes family for £7 million. In 2013, Mateschitz purchased a custom DeepFlight Super Falcon, an extreme $1.7 million submarine for guests at his Laucala Island resort in Fiji.

In late 2004, he bought the A1-Ring racing circuit, which had formerly hosted the Formula One Austrian Grand Prix, and renamed it the Red Bull Ring. The circuit re-opened in May 2011 and hosted a round of the 2011 Deutsche Tourenwagen Masters season. Although Mateschitz had stated that there were no plans for it to return to the Formula One calendar, in December 2012 Red Bull notified the Fédération Internationale de l'Automobile they would be open to hosting a Grand Prix. In July 2013, Red Bull announced the return of the Austrian Grand Prix to the Formula One World Championship in 2014. The race took place on 22 June 2014 and was won by Nico Rosberg, driving for Mercedes.