The biodiversity of potential leptospiral reservoir hosts is lower in urban than in rural environments. Previous data indicate the potential for bats to act as carriers of Leptospira in regions such as the Amazon of South America and in Australia. Yet, little is known about the contribution of bats to leptospirosis in urban environments in South America. This study aimed to test the hypothesis that bats infected with Leptospira are sources of leptospirosis transmission to humans in São Paulo City, Brazil. Six of 343 bats caught in different districts within the city of Sao Paulo (182 insectivorous, 161 frugivorous or nectarivorous) were polymerase chain reaction (PCR) positive for pathogenic Leptospira; no seropositive bats were found. That few renal carriers of Leptospira were found in the city of Sao Paulo suggests that bats are not important in the transmission of leptospirosis to humans in this, and possibly other urban settings.