With the advancement of nanotechnology, several nanoparticles have been synthesized as antimicrobial agents by utilizing biologically derived materials. In most cases, the materials used for the synthesis of nanoparticles from natural sources are extracts. Natural extracts contain a wide range of bioactive components, making it difficult to pinpoint the exact component responsible for nanoparticle synthesis. Furthermore, the bioactive component present in the extract changes according to numerous environmental factors. As a result, the current work intended to synthesize gold (AuNPs) and zinc oxide (ZnONPs) nanoparticles using pure phloroglucinol (PG). The synthesized PG-AuNPs and PG-ZnONPs were characterized using a UV–Vis absorption spectrophotometer, FTIR, DLS, FE-TEM, zeta potential, EDS, and energy-dispersive X-ray diffraction. The characterized PG-AuNPs and PG-ZnONPs have been employed to combat the pathogenesis of Pseudomonas aeruginosa. P. aeruginosa is recognized as one of the most prevalent pathogens responsible for the common cause of nosocomial infection in humans. Antimicrobial resistance in P. aeruginosa has been linked to the development of recalcitrant phenotypic characteristics, such as biofilm, which has been identified as one of the major obstacles to antimicrobial therapy. Furthermore, P. aeruginosa generates various virulence factors that are a major cause of chronic infection. These PG-AuNPs and PG-ZnONPs significantly inhibit early stage biofilm and eradicate mature biofilm. Furthermore, these NPs reduce P. aeruginosa virulence factors such as pyoverdine, pyocyanin, protease, rhamnolipid, and hemolytic capabilities. In addition, these NPs significantly reduce P. aeruginosa swarming, swimming, and twitching motility. PG-AuNPs and PG-ZnONPs can be used as control agents for infections caused by the biofilm-forming human pathogenic bacterium P. aeruginosa.