Nontuberculous mycobacteria (NTM) have recently emerged as important bacterial pathogens of animals and humans. Of particular concern is the high level of antimicrobial resistance displayed by these organisms, which complicates treatment and potential successful outcomes. Here, we evaluated the potential of Carlina acaulis L. as a source of novel anti-mycobacterial agents. Our goal was to measure the activity of aqueous, ethanol, and chloroform C. acaulis root extracts against 99 NTM strains. GC-MS spectroscopy analyses were performed to deliver qualitative and quantitative data on the composition of C. acaulis extract. In our study, we have shown for the first time the activity of C. acaulis extracts against NTM. The highest activity was exhibited by the chloroform extract, which inhibited the growth of more than 90% of the strains at the dose of 100 μg/mL (MIC90 = 100 μg/mL). The results of the GC-MS analysis of the C. acaulis chloroform extract contributed to the identification of 37 compounds, with carlina oxide as the most representative compound (69.52%) followed by 3,4-dihydro-2H-phenanthren- -1-one (6.54%) and stigmast-5-en-3-ol (4.14%). Our results indicate that C. acaulis chloroform and ethanol extracts have potential for treatment of NTM infections and that this plant contains anti-mycobacterial compounds.