UDP-3-O-[R-3-hydroxymyristoyl]-GlcNAc deacetylase enzyme of Pseudomonas aeruginosa is an interesting target for development of anti-infective drugs against this gram-negative bacterium. Many segregated studies analyzing the P. aeruginosa UDP-3-O-[R-3-hydroxymyristoyl]-GlcNAc deacetylase and its inhibitors have been reported in the recent past. In the present study, an attempt has been made to integrate this knowledge for the development of an effective multilayer screening approach. Eventually, an extensive chemical space was screened to filter out three potential P. aeruginosa UDP-3-O-[R-3-hydroxymyristoyl]-GlcNAc deacetylase inhibitors.