The referee pointed to the spot after the 24-year-old Argentine World Player of the Year was felled by defender Marian Cisovsky and he struck the ball into the net to the goalkeeper's right to become only the second Barca player to reach the mark.
Messi, top scorer in Europe's elite club competition the past three seasons, then added a second and third in stoppage-time in each half to complete a 14th career hat-trick on what was his 286th appearance according to Infostrada.
He also scored three in Saturday's 5-0 La Liga win at home to Real Mallorca and Tuesday's goals helped put holders Barca through to the Champions League last 16 with two games to spare.
"I am happy but more than anything for the result," Messi said in a pitchside interview with Spanish television, adding that he was not thinking about catching the Catalan club's record marksman Cesar Rodriguez, who scored 235.
"It's not my goal, far from it. The important thing was to win tonight and get through to the last 16.
"My aim is simply to keep going in the same way, to keep helping the team with my goals and together we will achieve some great things."
CLUB BEST
Messi took second spot on the list of Barcelona's record goal scorers from Hungarian Ladislau Kubala last month and is fast closing in on Cesar, who has held the record since the 1950s.
Barca goalkeeper Victor Valdes also wrote his name into the record books on Tuesday when he set a new club best for minutes unbeaten.
The 29-year-old has not conceded a goal in 877 minutes, beating a mark of 824 set in the 1972/73 season by Miguel Reina, father of Liverpool keeper and Valdes's Spain team mate Pepe Reina.
It is also the first time Barca have gone nine games without conceding. The last team to score against them were Valencia in La Liga on September 21.
