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This study was aimed to evaluate the antibacterial and biofilm inhibitory activity of Penicillium chrysogenum-derived silver nanoparticles (AgNPs) against the standard and pathogenic Acinetobacter baumannii using a 96-well microtiter plate-based method. The AgNPs were characterized by using UV–Vis, TEM, AFM, XRD, DLS, Zeta potential, and FT-IR. The nanoparticles (NPs) were fabricated with a spherical shape and an average hydrodynamic diameter of 48.2 nm. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of AgNPs were found to be 4 and 32 µg/mL respectively, whereas the MIC and MBC of tetracycline were found to be 1024 and 8192 µg/mL against A. baumannii (ATCC 19606). The AgNPs and tetracycline represented considerable biofilm inhibitory activity against both the standard and pathogenic A. baumannii at the studied concentrations. However, the AgNPs depicted higher potency to inhibit the process of biofilm formation of some pathogenic A. baumannii species compared to tetracycline. The AgNPs at the concentration of 0.5*MIC (2 µg/mL) inhibited above 90% biofilm inhibition, whereas tetracycline reached 90% biofilm inhibition at the concentration of 4*MIC (4096 µg/mL) against A. baumannii (ATCC 19606). However, further studies are required to evaluate the biofilm inhibitory efficacy of biogenic AgNPs in vivo. [ABSTRACT FROM AUTHOR]