Second-half substitute Matt Clare grabbed the only goal of the game; however Evan Newton in the home goal was the star of the show as he made a series of impressive saves to deny the visitors.
The substitute stopper denied Kevin Davies, Chung-Yong Lee and Robbie Blake in quick succession before Tampa Bay grabbed the winner on 65 minutes, with a tidy first-time finish from Clare.
Martin Petrov also hit a post for Bolton, who fielded a total of 21 players throughout the 90 minutes having started with eight of the first XI from the clash against Manchester City in the final game of last season.
"It was a fantastic experience," Owen Coyle said.
"We knew it would be a tough game, we knew it would be demanding physically.
"We thoroughly enjoyed the exercise. We could have score six or seven goals, there's no doubt about that and ultimately Tampa scored a very good goal so credit to them."