Research concerning leptospirosis in donkeys and mules has been neglected around the world. Therefore, the aim of this study was to investigate the epidemiological situation of the prevalence of anti-Leptospira spp. antibodies in donkeys and mules from the state of Minas Gerais, Brazil. Blood serum samples were collected from 180 animals (109 donkeys and 71 mules) in two rural properties from the state of Minas Gerais, Brazil, and then submitted to a microscopic agglutination test (MAT). Urea and creatinine values were also quantified. Epidemiological variables such as age, breeding system, contact with other animal species, source of water and food, vaccination against leptospirosis, presence of reproductive alterations, and rodent control were also investigated. From 180 samples collected, 39 (21.67%) showed positive results in the MAT, at a dilution ≥ 1:100. Some animals were reactive for more than one serovar. The serovar Tarassovi was the most frequent (14.07%), followed by Hardjo (11.85%) and Wolffi (11.11%). There was a statistically significant difference between animals from 0 to 3 years of age reactive in the MAT in comparison to the other age groups. Most of the animals had urea and creatinine concentrations within the acceptable reference limit; however, there was a significant increase in creatinine levels in some of the test animals. The studied properties showed differences in some epidemiological aspects such as vaccination of the animals, presence of reproductive problems in the herd, and rodent control. Such aspects pointed as risk factors that may influence the frequency of positive serological results in property 1. The present study demonstrated that the prevalence of leptospirosis in donkeys and mules is high and several serovars are being maintained by these animals, representing a potential public health risk.