Nanoparticle synthesis using biosurfactants is currently a popular and environmentally friendly method for improving both particle synthesis and stabilization. Zinc oxide nanoparticles (ZnO NPs) have found extensive applications in the field of biomedicine. The present study focused on isolating microorganisms that produce biosurfactants from soil contaminated with oil. The isolated microorganisms were identified through molecular identification of 16s r-RNA gene sequencing and characterization as Bacillus pseudomycoides. We determined biosurfactant production using various assays such as hemolysis assay, emulsification index, oil spreading tests, and drop-collapsing assays. This investigation confirms that isolated microorganisms possess biosurfactant properties. We utilized a biosurfactant extract from the B. pseudomycoides strain for the synthesis of ZnO NPs. Advanced techniques such as UV-vis spectroscopy, XRD, FTIR, SEM, and Edax were employed to ensure accurate characterization. Furthermore, the biosurfactant-mediated ZnO NPs effectively killed a few bacterial pathogens, such as Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumonia. They were also effective against fungal pathogens, such as Aspergillus niger, Aspergillus flavus, and Fusarium oxysporum. This research highlights the impressive effectiveness of newly synthesized biosurfactant-mediated ZnO against clinical isolates. This research suggests that the biosurfactant extract from B. pseudomycoides could be used to make ZnO nanoparticles that could be useful for treating harmful infections.