Tuberculosis (TB) remains a major threat to human health worldwide. The increasing incidence of non-tuberculous mycobacterial infections and particularly those produced by Mycobacterium avium has emphasized the need to develop new drugs. Additionally, high levels of natural drug resistance in non-tuberculous mycobacteria (NTM) and the emergence of multidrug-resistant (MDR) TB is of great concern. Antimicrobial peptides (AMPs) are antibiotics with broad-spectrum antimicrobial activity. The objective was to assess the activity of AMPs against Mycobacterium tuberculosis and M. avium clinical isolates. MICs were determined using microtitre plates and the resazurin assay. Mastoparan and melittin showed the greatest activity against M. tuberculosis, while indolicidin had the lowest MIC against M. avium. In conclusion, AMPs could be alternatives for the treatment of mycobacterial infections. Further investigation of AMPs' activity in combination and associated with conventional antibiotics and their loading into drug-delivery systems could lead to their use in clinical practice.