Over the last 30 years, the number of invasive turtle species living in the wild has significantly increased in Poland. This proliferation carries many threats, which mainly include the displacement of native species of animals from their natural habitats. Turtles can also be reservoirs for pathogens, including bacteria from the Mycobacterium genus. In order to confirm or rule out the presence of acid-fast mycobacteria in the population of invasive turtle species, samples from carapace, plastron, internal organs and mouth cavity swabs from 125 animals were tested. Twenty-eight mycobacterial strains were isolated in culture, which were classified as atypical following multiplex-PCR reactions. The GenoType Mycobacterium Common Mycobacteria (CM) test, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, PCR-restriction fragment length polymorphism (PRA)-hsp65 and DNA sequencing were used to identify the species of isolates. Of the 28 strains, 11 were identified as M. fortuitum, 10 as M. chelonae, 3 as M. avium ssp. avium, 2 as M. nonchromogenicum and 1 each of M. neoaurum and M. scrofulaceum. The results of the research will also strengthen the understanding that these animals can be vectors for pathogens when living in the wild.