Mycobacterium is one of the most dangerous pathogens of both animals and humans. Bovine tuberculosis (BTB) is a disease caused by mycobacteria belonging to the Mycobacterium tuberculosis complex (MTBC), which spreads mainly among domestic cattle but also to mammals other than cattle. The transmission of MTBC between different species requires research and epidemiological investigations to control its spread. When multiple species are a reservoir of infection, it poses a significant public health and veterinary concern. In this study, the diagnosis of alpaca, cattle, horses, dogs, a sheep and a cat from one farm suspected of bovine tuberculosis was performed. The animals (except for one horse, the dogs and the cat) were euthanised after the intradermal tuberculin tests. Mycobacterial isolation from animal tissue samples was performed. The obtained Mycobacterium strains were genotyped using spoligotyping and mycobacterial interspersed repetitive unit–variable number tandem repeat (MIRU-VNTR) methods. The isolates from a horse, two cows, a sheep and an alpaca were classified as Mycobacterium (M.) bovis. The single M. bovis spoligotype SB0666 pattern was isolated, and the MIRU-VNTR results presented the same 222632237401435 patterns. The molecular investigation uncovered information on the relationship of Mycobacterium bovis.