Background Leptospirosis is a significant zoonosis in tropical regions, where poor sanitation and favourable climate aid its spread. Synanthropic animals such as the cane toad (Rhinella jimi), which share environments with both people and wild and domestic animals, may harbour Leptospira and contribute to urban and rural transmission cycles.Methods This study evaluated the serological and molecular presence of Leptospira spp. in R. jimi collected from urban and rural areas in Brazil's semi-arid region. Twenty-five toads were captured, euthanased and sampled. Blood was analysed using the microscopic agglutination test, and urine and kidney tissues were tested via polymerase chain reaction (PCR) targeting the LipL32 gene.Results Six toads (24%) were seropositive for Leptospira, with the identified serogroups being Australis (n = 3), Icterohaemorrhagiae (n = 2) and Pomona (n = 1). PCR detected Leptospira DNA in 11 animals (44%), mainly from urban locations.Limitations The study's small sample size, although ethically justified, represents a limitation for broader epidemiological inferences. Furthermore, due to the lack of complete data on the animals' prior movements, the relationship between infection and geographical origin could not be fully determined.Conclusion The presence of antibodies and leptospiral DNA indicates that R. jimi may act as a non-conventional host in Leptospira transmission, posing a potential public health risk.