Recently, the nitro-substituted bisquaternary bisnaphthalimides were reported to have substantial anti-infective activity against Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA). Here, we selected resistant S. aureus clones by cultivation in increasing concentrations of the most active compound, MT02. Interestingly, MT02-resistant variants induced a diffusible red color of the broth. Chromatographic and spectroscopic investigations revealed a stepwise reduction of the bisquaternary bisnaphthalimides' nitro groups to amino groups. The corresponding derivatives were completely inactive against staphylococci. RNA sequencing experiments revealed a strong overexpression of a novel oxidoreductase in MT02-resistant strains. Deletion mutants of this enzyme did not produce the red color and were not able to develop resistance against bisquaternary bisnaphthalimides. Biochemical reactions confirmed an NADH-dependent deactivation of the nitro-substituted compounds. Thus, this is the first report of a nitroreductase-based antibiotic resistance mechanism in the human pathogen S. aureus.