The O-acetylation of peptidoglycan (PG) is now known to occur in 53 species, including numerous human pathogens such as, Staphylococcus aureus, Bacillus anthracis, species of Enterococcus, Campylobacter jejuni, Helicobacter pylori, Neisseria gonorrhoeae and N. meningitidis. This modification, which occurs at the C-6 hydroxyl of N-acetylmuramoyl residues within PG, serves to regulate autolytic activity during PG metabolism and contributes to pathogenesis and persistence within a host. O-Acetylpeptidoglycan esterase (Ape) was recently discovered as an enzyme responsible for the removal of O-acetyl groups from PG, thus permitting the continued maintenance and metabolism of the sacculus. Recombinant Ape1 from N. gonorrhoeae was purified to apparent homogeneity and optimal storage conditions for the enzyme were determined. Using 4-methylumbelliferyl acetate as substrate, a fluorogenic assay amenable for the high-throughput screening for potential inhibitors was developed and Ape1 was screened against a subset of compounds of the Canadian Compound Collection. The overall Z′ score for the screen was 0.62, indicative of a well-suited assay with a sufficient signal window, and the threshold was set at 65 %. After eliminating a number of false-positives, seven compounds were identified as true inhibitors of Ape1, the first to be identified for this class of enzyme. Dose–response curves were generated leading to the identification of five of these compounds with IC50 values ranging between 0.3 and 23 μM. Of these, purpurin was selected for further analysis and it was found to inhibit the growth of both Gram-positive and Gram-negative bacteria that produce both O-acetylated PG and Ape.