The Trotters arrived in Spain holding a one-goal lead from the first leg, El-Hadji Diouf having netted the only goal of the game in the Reebok Stadium, but the Senegal forward had to settle for a place on the bench.
A nervy opening saw Joey O'Brien cautioned for a foul on former Arsenal forward Jose Antonio Reyes, before Bolton almost struck a decisive early blow.
Stelios Giannakopoulos was well placed 12 yards from goal, but his shot was tipped over the crossbar by Italian goalkeeper Christian Abbiati.
Atletico soon hit back, with Mariano Pernia firing wide from 20 yards, before Jussi Jaaskelainen had to make a smart save from Maxi Rodriguez's close-range effort.
Former Premier League pair Diego Forlan and Luis Garcia both failed to test Jaaskelainen further, but Bolton were now under the cosh and Forlan again went close just before the interval.
After the break the home side continued to dominate, as Pernia and Garcia again threatened Jaaskelainen's goal.
Just before the hour Bolton boss Gary Megson rang the changes, introducing first-leg goalscorer Diouf and Ivan Campo for Gavin McCann and Giannakopoulos.
Cleber Santana then shot wide for Atletico from 12 yards out, as the Spaniards continued to push forward looking for the goal they needed to level up the aggregate score.
Reyes fired a shot wide from the edge of the area following a corner 16 minutes from time, before both Diouf and Kevin Davies were cautioned as Bolton desperately hung on to the narrowest of advantages.
Miguel fired the last chance of the game wide of target and Bolton were left to celebrate their achievement in reaching the last 16 of the competition.