The objective of this study was to find molecules with anti-mycobacterial activity from a natural compounds library, investigate their mechanisms of resistance, and assess their synergy with antibiotics. We screened a library of 2582 natural compounds with Mycobacterium aurum with the aim of identifying molecules with anti-mycobacterial activity. The hits with the lowest MICs in M. aurum were also tested for their antimicrobial activity in other mycobacterial species including M. tuberculosis complex strains. The chequerboard titration assay was chosen for determining drug interactions in vitro. Spontaneous resistant mutants were isolated and their whole genome sequences compared to wild type and resistant mutants to identify resistance mechanisms. We found that ionophores show anti-mycobacterial activity in vitro. Resistance mechanism to ionophores is mediated by the MmpL5-MmpS5 transporter overexpression. Ionophore A23187 enhanced beta-lactam activity in M. tuberculosis infected macrophage. It will help in the investigation of new drug combinations against bacterial infections including tuberculosis.