Nick Solak singled home Logan Taylor with one out in the bottom of the ninth inning and freshman Blake Tiberi singled home Mike White - who had reached on an infield hit - in the bottom for the 10th inning for a 2-1 win by the Cardinals.
It was the fifth walk-off win for the Cardinals. The game was played before a regular-season record crowd of 5,056 at Jim Patterson Stadium.
Zach Burdi worked a perfect ninth inning to get the victory for the Cardinals. Sean Leland had worked seven innings, allowing six hits and one run, in his third start of the season. Robert Strader and Lincoln Henzman worked an inning apiece.
But while the pitching was solid, the Cardinals didn't get a leadoff runner on base until the ninth inning and had just three base runners in the first eight innings.
Pinch hitter Danny Rosenbaum walked to start the ninth before pinch runner Logan Taylor was moved to second on a sacrifice by Sutton Whiting. Taylor then stole third and was driven home by Solak, who extended his hitting streak to 20 games.
White then started the 10th inning with an infield single and moved to second on a throwing error. Tiberi, a pinch hitter, then drove a 2-2 pitch into center field.
Louisville (35-10) will play at Clemson on Saturday, Sunday and Monday. The Cardinals are 19-2 in ACC play and ranked as high as No. 3 nationally.