This study was aimed to isolate and identify Mycobacterium bovis and non-tuberculous mycobacteria (NTM) species in raw milk samples from cattles and to compare the diagnostic performance of the methods used for that purpose. A total of 145 raw milk samples from cattles were collected from five villages in Mersin province (located on Mediterrenean region of Turkey) between April and June 2008. Presence of mycobacteria was investigated by Ehrlich Ziehl Neelsen (EZN) staining method, culture in Lowenstein-Jensen (LJ) medium and polymerase chain reaction (PCR). Only 1 (0.7%, 1/145) raw milk sample was found to be acid fast bacilli (AFB) positive with EZN staining. Eleven (7.6%) samples were positive by culture and mycobacterial DNA was detected in 6 (4.1%) samples by PCR. Mycobacterium was isolated from both creamy and pellet layer of a culture positive sample. Identification was carried out with conventional biochemical tests, PCR-Restriction Fragment Length Polymorphisms (PCR-RFLP) and spoligotyping (spacer oligonucleotide typing) methods. One isolate was identified as Mycobacterium tuberculosis complex (MTC) and 11 isolates were identified as NTM out of 12 isolates those were isolated from culture. According to PCR-RFLP analysis of these 11 NTM isolates, 6 (54.5%) were Mycobacterium genavense, 2 (18.2%) were Mycobacterium simiae, 2 (18.2%) were Mycobacterium szulgai and 1 (9.1%) was Mycobacterium fortuitum. MTC isolate was identified as M.bovis by spoligotyping. According to the results of our study, both pellet and creamy layers from raw milk samples should be cultured to selective LJ medium (without glycerol, with 0.4% sodium pyruvate) to improve the chance of isolation and must be incubated for up to eight weeks. In our region, NTM were isolated in 6.9% and M.bovis in 0.7% of the raw milk samples from cattles and this emphasized the risk of transmission of mycobacteria to man via direct contact or ingestion of unpasteurized milk products.