There is a global health crisis of antimicrobial resistance, responsible for over a million deaths annually. Mycobacterial infections are a major contributor to this crisis, causing more deaths than any other single infectious agent. Notably, the rise of multidrug-resistant (MDR), extensively drug-resistant (XDR), and totally drug-resistant (TDR) strains of has led to higher mortality rates and challenge all existing antibiotic regimens. Light-activated molecular nanomachines (MNMs) represent a promising class of broad-spectrum antimicrobial agents that could help counter this rise in antimicrobial resistance. Addressing a key knowledge gap, this study explores the mechanisms of action for MNMs in , a surrogate model for pathogenic mycobacteria. We show that fast-rotor MNMs significantly reduce bacterial viability, achieving up to 97 % reduction in with 30 minutes of light activation when compared to non-activated MNM ( < 0.0001, = 24.55), as determined by an unpaired -test. Using fluorescence and confocal microscopy, we also show the colocalization of MNM with as part of their mechanism of action. The ability to translate these observations to pathogenic mycobacteria was demonstrated by the ability of MNM to kill 93.5 % of with 5 minutes of light activation when compared to non-activated MNM ( < 0.0001, = 19.24). These findings suggest that MNMs have the potential to be innovative and sustainable antimicrobial agents for the treatment of pathogenic mycobacterial infections.