Leptospirosis has been widely reported in insular environments worldwide, characterizing a major public health threat. Although low genetic biodiversity is expected in these regions, the introduction of domestic and synanthropic mammals may contribute to the wider diversity leptospiral strains in insular settings. This study proposes a large-scale investigation of Leptospira infection in animals from Fernando de Noronha archipelago, Brazil. A total of 1,265 blood samples from domestic (n=682), synanthropic (n=133) and wild (n=450) animals were collected between 2007 and 2014, totaling 12 species. The presence of anti-Leptospira spp. antibodies was investigated by the Microscopic Agglutination Test (MAT) and kidney samples from synanthropic rodents were collected for the isolation of Leptospira spp. The leptospires recovered were further characterized by MAT with polyclonal antibodies, whole genome sequencing and Multilocus Sequence Typing (MLST). The MAT results revealed the presence of agglutinins in 90 samples (7.1%), and the most frequently found serogroup was Icterohaemorrhagiae (n=57) in practically all species included. Viable leptospires were recovered from one brown rat, and characterization revealed that the isolate belongs to L. interrogans serogroup Pyrogenes. This study stands as the most comprehensive investigation of Leptospira spp. infection in Fernando de Noronha archipelago, also providing the characterization of the first leptospiral strain ever isolated from an insular setting in Brazil. The results suggest that synanthropic rodents play a major role in the transmission of leptospirosis among wildlife and domestic species in the archipelago.