Samples were collected from 128 symptomatic humans, 83 dogs, 49 mice, and 20 rats (Rattus rattus: 16; Rattus norvegicus: 4) in neighborhoods where human leptospirosis have been reported within the principal sea-port city of Colombia. Seroprevalences were assessed against 19 pathogenic, 1 intermediate pathogenic, and 1 saprophytic Leptospira serogroups. Pathogenic Leptospira were confirmed using conventional Leptospira-specific polymerase chain-reaction and pulsed-field gel electrophoresis analysis was used for serovar identification. Seroprevalences of 20.4%, 12.5%, 25.0%, 22.9%, and 12.4% were obtained against one to seven different serogroups in mice, R. rattus, R. norvegicus, dogs, and humans, respectively. The DNA was confirmed to be from pathogenic Leptospira by detecting the lipL32 gene in 12.5%, 3.7%, and 0.03% of the R. rattus, dog, and human samples, respectively. The first genetically typed Colombian isolate was obtained from a rat and identified as Leptospira interrogans serovar Icterohaemorrhagiae/Copenhageni.