In 1977, he took over his family's pharmaceutical wholesaler in Naples and turned it into Alliance Santé, a Franco-Italian pharmaceutical wholesale group. In 1997, it merged with Alliance UniChem, and he joined its board of Directors. From 2001 to 2004, he served as its CEO. He served as deputy chairman, and later as chairman.
He has also been the romantic partner of Ornella Barra for over 30 years. They met in 1985, as she was just beginning her career. She is 13 years his junior and shares his Monaco residence and work schedule, acting as co-chief operating officer of Walgreens Boots Alliance.
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".
On 2 February, Walgreens Boots Alliance stated, "The comments made by Stefano Pessina were a small part of a much larger conversation and have been taken out of context ... Stefano Pessina was expressing his personal views only and is not campaigning against Ed Miliband or the Labour Party." On 6 February, Allister Heath, deputy Editor of The Telegraph, described the "attacks" by Labour and Liberal Democrat politicians on Pessina, "one of the world’s greatest entrepreneurs", as being "as nonsensical as they were indefensible".