This case report describes a case of leptospirosis in a pampas deer (Ozotoceros bezoarticus) from Southern Brazil which was attributed to four serotypes of Leptospira interrogans. Serum was prepared and analyzed using a panel of eight serotypes. The animal showed clinical signs of leptospirosis and was positive for four serotypes (Leptospira hardjo, Leptospira wolffi, Leptospira butembo, and Leptospira bratislava), characterizing a mixed infection. As far as we know, this is the first report of L. butembo and L. bratislava in O. bezoarticus. This suggests that wild pampas deer can be a source of (mixed infection) leptospirosis.