Non-tuberculous mycobacteria (NTM), particularly mycolactone producing mycobacteria (MPM), are bacteria found in aquatic environments causing skin diseases in humans like Buruli ulcer (BU). Although the causative agent for BU, Mycobacterium ulcerans has been identified and associated with slow-moving water bodies, the real transmission route is still unknown. This study aimed to characterize MPMs from environmental aquatic samples collected in a BU non-endemic community, Adiopodoumé, in Côte d’Ivoire. Sixty samples were collected in four types of matrices (plant biofilms, water filtrate residues, plant detritus and soils) from three water bodies frequently used by the population. Using conventional polymerase chain reaction (PCR), MPMs were screened for the 16S ribosomal RNA (rRNA) mycobacterial gene, the IS2404 insertion sequence, and MPM enoyl reductase (ER) gene. Variable Number Tandem Repeat (VNTR) typing with loci 6, 19, mycobacterial interspersed repetitive unit 1 (MIRU1) and sequence type 1(ST1) was performed to discriminate between different MPMs. Our findings showed 66.7%, 57.5% and 43.5% of positivity respectively for 16S rRNA, IS2404 and ER. MPM discrimination using VNTR typing did not show any positivity and therefore did not allow precise MPM distinction. Nevertheless, the observed contamination of some water bodies in a BU non-endemic community by MPMs suggests the possibility of pathogen dissemination and transmission to humans. These aquatic environments could also serve as reservoirs that should be considered during control and prevention strategies.