Few studies have addressed Leptospira seroprevalence and risk factors in urban populations in Colombia. This study aimed to determine seroprevalence and factors associated with Leptospira infection in inhabitants of an urban district of Cali, Colombia. We collected sociodemographic and environmental data, as well as blood samples, from 353 subjects selected through a multistage cluster sampling design. We performed microagglutination test for the eight main Leptospira serogroups circulating in the region, considering a cut-off titer of ≥ 1:100. Most participants were female (226, 64.8%), with mean age 41.4 years, and 89 (32.6%) lived in low-low socioeconomic stratum (SES-1). Overall seroprevalence was 12.2% (95%CI: 10.3%-14.4%). Factors associated with Leptospira infection were SES 1, older age, single marital status, ethnic groups (Afro-Colombian and white/mestizo), school students, absence of toilet, barefoot walking, travel outside Cali in the previous month, and absence of skin and mucous-membrane lesions in the previous month. Our study suggests domestic and peridomiciliary transmission of Leptospira likely related to activities of daily living and inadequate environmental conditions. SES-1 is a major factor associated with Leptospira infection (adjusted OR = 4.08; 95%CI: 2.54-6.53; p < 0.001), suggesting that social and environmental conditions are key elements for endemicity of Leptospira infection in the study area. Epidemiological surveillance, improvement of environmental and sanitary conditions in various SES-1 areas, and community educational campaigns are recommended.