Mycobacteria are ubiquitous microorganisms and may be found in many different environments, either associated or not to diseases. Culture-independent molecular techniques brought new perspectives for research regarding soil microorganisms, which is relevant to microbiologists and industry. Therefore, new research is necessary to broaden our knowledge about ecology and physiology of mycobacteria in extreme environments. The aim of this study was to verify the presence of mycobacteria in Antarctica soil samples and to standardize molecular and culture-dependent techniques to their isolation. Soil DNA was extracted and submitted to denaturing gradient gel electrophoresis (DGGE), rrs mycobacterial gene amplification and partial sequencing. Thereby, 10 colonies morphologically similar to mycobacteria were isolated from 10 different samples, resulting in 15 DNA sequences where Mycobacterium neglectum and Mycobacterium kumamotonense were identified, suggesting that Mycobacterium terrae complex representatives are ubiquitous in the region. This study proposes a simple method for mycobacteria cultivation from soil samples and shows that ornithogenic soils can be the source of organic matter and low pH, modulating the mycobacterial Antarctica diversity. Further research aiming to explore mycobacterial diversity in soil are needed to increase knowledge and better explore their evolution, their relation with animal and human species and their potential biotechnological use.