Leptospira spp., a zoonotic agent relevant for public health, occurs frequently in tropical regions. The aquatic environment represents a viable survival and transmission pathway. This study aimed to investigate the presence of anti-Leptospira spp. antibodies in Phrynops geoffroanus (Geoffroy's side-necked turtle) serum samples using the microagglutination test (MAT), and Leptospira spp. in gastric and cloacal lavage samples using the polymerase chain reaction (PCR) technique. Antibodies against nine different Leptospira spp. serovars were detected in 45.45% (30/66) of the serum samples. Specific amplification of Leptospira spp. genomic material (331bp) was observed in 16.67% (11/66) of the samples. In conclusion, these freshwater testudines host Leptospira spp. and eliminate them. This situation may represent a risk to public health, especially to people who use urban streams for fishing and recreational activities. Additionally, we described some Leptospira spp. serovars, not yet reported in testudines, detected here in P. geoffroanus.