NB-003 and NB-003 gel formulations are oil-in-water nanoemulsions designed for use in bacterial infections. In vitro susceptibility of Propionibacterium acnes to NB-003 formulations and comparator drugs was evaluated. Both NB-003 formulations were bactericidal against all P. acnes isolates, including those that were erythromycin, clindamycin, and/or tetracycline resistant. In the absence of sebum, the MIC(90)s/minimum bactericidal concentrations (MBC(90)s) for NB-003, NB-003 gel, salicylic acid (SA), and benzoyl peroxide (BPO) were 0.5/2.0, 1.0/2.0, 1,000/2,000, and 50/200 μg/ml, respectively. In the presence of 50% sebum, the MIC(90)s/MBC(90)s of NB003 and BPOs increased to 128/1,024 and 400/1,600 μg/ml, respectively. The MIC(90)s/MBC(90)s of SA were not significantly impacted by the presence of sebum. A reduction in the MBC(90)s for NB-003 and BPO was observed when 2% SA or 0.5% BPO was integrated into the formulation, resulting in MIC(90)s/MBC(90)s of 128/256 μg/ml for NB003 and 214/428 μg/ml for BPO. The addition of EDTA enhanced the in vitro efficacy of 0.5% NB-003 in the presence or absence of 25% sebum. The addition of 5 mM EDTA to each well of the microtiter plate resulted in a >16- and >256-fold decrease in MIC(90) and MBC(90), yielding a more potent MIC(90)/MBC(90) of ≤1/<1 μg/ml. The kinetics of bactericidal activity of NB-003 against P. acnes were compared to those of a commercially available product of BPO. Electron micrographs of P. acnes treated with NB-003 showed complete disruption of bacteria. Assessment of spontaneous resistance of P. acnes revealed no stably resistant mutant strains.