Tuberculosis is the leading cause of death due to a single infectious agent in the world and the emergence of multidrug-resistant strains prompted us to develop new drugs with novel targets and mechanism. Here, we screened a natural antibiotics library with Mycobacterium smegmatis membrane-bound dehydrogenases and identified polymyxin B (cationic decapeptide) and nanaomycin A (naphtoquinone derivative) as inhibitors of alternative NADH dehydrogenase [50% inhibitory concentration (IC(50)) values of 1.6 and 31 microg/ml, respectively] and malate: quinone oxidoreductase (IC(50) values of 4.2 and 49 microg/ml, respectively). Kinetic analysis on inhibition by polymyxin B showed that the primary site of action was the quinone-binding site. Because of the similarity in K(m) value for ubiquinone-1 and inhibitor sensitivity, we examined amino acid sequences of actinobacterial enzymes and found possible binding sites for L-malate and quinones. Proposed mechanisms of polymyxin B and nanaomycin A for the bacteriocidal activity were the destruction of bacterial membranes and production of reactive oxygen species, respectively, while this study revealed their inhibitory activity on bacterial membrane-bound dehydrogenases. Screening of the library with bacterial respiratory enzymes resulted in unprecedented findings, so we are hoping that continuing efforts could identify lead compounds for new drugs targeting to mycobacterial respiratory enzymes.