The objective of this study was to analyze the eco-epidemiological aspects of Leptospira seroprevalence and seroincidence and its associated factors in two municipalities of northwest Colombia. A prospective study was performed in rural areas of Urabá, Antioquia, Colombia. The study enrolled 597 people between November 2015 and January 2016, of which 274 were followed up 1 year later. Serologic testing was performed by a microscopic agglutination. The outcomes were seroprevalent and seroincident cases, and the primary exposure was an outdoor occupation. A binary and mixed-effect multinomial logistic regression model was used to estimate factors associated with seroprevalent or seroincident cases of Leptospira infection. The overall Leptospira seroprevalence was 27.81% (95% confidence interval: 23.62-32.49), and the overall cumulative seroincidence for Leptospira was 14.60% (95% confidence interval: 10.33-20.23). Multivariable analysis showed that factors associated with L. interrogans serogroups seropositivity were outdoor occupation, male gender, older age, the presence of dirt floor in the household, and the presence of piglets and opossums. It also showed that factors associated with other Leptospira species serogroups were the presence of pit latrines and of turkeys. In addition, the multivariable model of seroincident cases of L. interrogans serogroups evidenced outdoor occupations, the presence of rats, and corn cultivation as risk factors. Likewise, the multivariable model for seroincident cases of other Leptospira species showed that the presence of hunting canines and cassava cultivation were risk factors. We found specific factors associated with the transmission of Leptospira serogroups contribute to the understanding of the epidemiology of Leptospira infection in rural areas of Urabá, Colombia.