Leptospirosis is a bacterial zoonosis caused by pathogenic species from the genus Leptospira. Infection mostly occurs through indirect contact with environmental water contaminated with the urine of reservoir animals. Information on the circulation of leptospirosis in West Africa, as well as its potential reservoir hosts, is limited. Therefore, we carried out trapping surveys in the Guinean forest in November 2022, and samples were collected from 42 micromammals. The animals were both morphologically and genetically identified. The lungs and kidneys were screened for Leptospira using Lfb1-gene-targeting real-time PCR, and positive samples were genotyped based on the polymorphic Lfb1 gene. Leptospira species were detected in the kidneys of three micromammals: Mastomys natalensis, Lophuromys sikapusi, and Rattus rattus. Leptospira borgpetersenii was identified in Rattus rattus and Mastomys natalensis that were captured in two different villages. The phylogenetic analysis indicated that this subspecies had previously been detected in one patient in Mayotte, but the reservoir was not identified. A new subspecies of Leptospira kirschneri was isolated in Lophuromys sikapusi from the same village as the Mastomys natalensis positive for L. borgpetersenii. The high diversity of both the reservoirs and Leptospira species in the Guinean forest indicates that we should study other natural regions and reinforce communities’ awareness of Leptospira infection risks in Guinea.