Senegal international Diouf struck 16 minutes from time at the Reebok Stadium, soon after the visitors had had substitute Sergio Aguero sent off.
Trotters boss Gary Megson handed European debuts to January recruits Gary Cahill and Matt Taylor and the latter threatened three times in the opening ten minutes, being denied on each occasion by Italian goalkeeper Christian Abbiati.
However, 12 minutes in Bolton almost fell behind as Antonio Lopez met Jose Antonio Reyes' free-kick and Jussi Jaaskelainen made a superb one-handed save from the defender's header.
Former Manchester United frontman Diego Forlan almost capitalised on a defensive mix-up on the half-hour, but Andy O'Brien recovered to avert the danger.
Forlan was the first to threaten after the interval, with his shot from the edge of the area flying just a couple of feet wide of Jaaskelainen's goal.
Atletico seemed to have the game under control, but things changed 16 minutes from time when Aguero reacted to a challenge from Taylor and was dismissed for allegedly spitting at the Bolton man.
Within a couple of minutes Bolton took the lead as Diouf capitalised on good approach play by Stelios Giannakopoulos and ex-Portsmouth wing-back Taylor to break the deadlock.
Atletico pushed forward as time ticked away, but they failed to come close to grabbing an equaliser, leaving Megson's side to celebrate a famous victory on home turf.