The Gram-negative human pathogenNeisseria gonorrhoeaehas progressively developed resistance to antibiotic monotherapies, and recent failures of dual-drug therapy have heightened concerns that strains resistant to all available antibiotics will begin circulating globally. Targeting bacterial cell wall assembly has historically been effective at treating infections withN. gonorrhoeae, but as the effectiveness of β-lactams (including cephalosporins) is challenged by increasing resistance, research has expanded into compounds that target the numerous other enzymes with roles in peptidoglycan metabolism. One example is the dithiazoline compound JNJ-853346 (DTZ), which inhibits the activity of anEscherichia coliserine proteasel,d-carboxypeptidase (LdcA). Recently, the characterization of an LdcA homolog inN. gonorrhoeaerevealed localization and activity differences from the characterizedE. coliLdcA, prompting us to explore the effectiveness of DTZ againstN. gonorrhoeae. We found that DTZ is effective at inhibitingN. gonorrhoeaein all growth phases, unlike the specific stationary-phase inhibition seen inE. coli. Surprisingly, DTZ does not inhibit gonococcal LdcA enzyme activity, and DTZ sensitivity is not significantly decreased inldcAmutants. While effective against numerousN. gonorrhoeaestrains, including recent multidrug-resistant isolates, DTZ is much less effective at inhibiting growth of the commensal speciesLactobacillus gasseri. DTZ treatment during coinfections of epithelial cells resulted in significant lowering of gonococcal burden and interleukin-8 secretion without significantly impacting recovery of viableL. gasseri. This selective toxicity presents a possible pathway for the use of DTZ as an effective antigonococcal agent at concentrations that do not impact vaginal commensals.