In 1977, Pessina took over his family’s pharmaceutical wholesaler (possibly Petrone Group) in Naples, Italy, and turned it into Alliance Santé, a Franco-Italian pharmaceutical wholesale group. In 1997, it merged with Alliance UniChem. From 2001 to 2004, he served as its CEO. He served as deputy chairman, and later as chairman. The company merged with Boots Group PLC in 2006 and was taken private in July 2007. Pessina was chairman of Alliance Boots from 2007 to 2014 and currently serves as Executive Vice Chairman and Chief Executive Officer. Pessina is a director of Walgreens Boots Alliance. He is a director of the Consumer Goods Forum.

In a 31 January 2015 interview for The Sunday Telegraph, Pessina commented on the prospect of Britain’s Labour Party winning the UK general election in May 2015, “If they acted as they speak, it would be a catastrophe … The problem is would they act that way or not? One thing is to threaten and to shout but it is completely different to be in charge and to manage the country day-to-day.” He described Labour’s business policies as “not helpful for business, not helpful for the country and in the end it probably won’t be helpful for them”. The Sunday Telegraph noted that Pessina “declined to elaborate on which specific policies he disliked”.

In July 2020, it was announced that Pessina would step down as CEO of WBA to take a “less hands on role” and replace Jim Skinner as executive chairman.