Leptospirosis is the most widespread zoonosis in the world. In northern Botswana, humans live in close proximity to a diversity of wildlife and peridomestic rodents and may be exposed to a variety of zoonotic pathogens. Little is known regarding the occurrence and epidemiology of L. interrogans in Africa despite the recognized global importance of this zoonotic disease and the threat it poses to public health. In Botswana, banded mongooses (Mungos mungo) live in close proximity to humans across protected and unprotected landscapes and may be a useful sentinel species for assessing the occurrence of zoonotic organisms, such as L. interrogans. We utilized PCR to screen banded mongoose kidneys for leptospiral DNA and identified 41.5% prevalence of renal carriage of L. interrogans (exact binomial 95% CI 27.7–56.7%, n = 41). Renal carriage was also detected in one Selous' mongoose (Paracynictis selousi). This is the first published confirmation of carriage of L. interrogans in either species. This is also the first report of L. interrogans occurrence in northern Botswana and the only report of this organism in a wildlife host in the country. Pathogenic Leptospira are usually transmitted indirectly to humans through soil or water contaminated with infected urine. Other avenues, such as direct contact between humans and wildlife, as well as consumption of mongooses and other wildlife as bushmeat, may pose additional exposure risk and must be considered in public health management of this newly identified zoonotic disease threat. There is a critical need to characterize host species involvement and pathogen transmission dynamics, including human–wildlife interactions that may increase human exposure potential and infection risk. We recommend that public health strategy be modified to include sensitization of medical practitioners to the presence of L. interrogans in the region, the potential for human infection, and implementation of clinical screening. This study illustrates the need for increased focus on neglected zoonotic diseases as they present an important threat to public health.