The emergence of resistance by biofilm-forming bacteria has reached alarming and dangerous levels that threaten human civilization. The current study sought to investigate the antibiofilm potential of green-synthesized silver nanoparticles, mediated by a new Streptomyces strain. Zeta potential, transmission electron microscopy (TEM), and UV-Vis spectroscopy were used to analyze the biosynthesized AgNPs. Results revealed that silver nanoparticles had a size of (5.55 and 45.00 nm) nm and a spherical shape, with surface plasmon resonance (SPR) absorption at 400–460 nm in the UV-vis spectra establishing the formation of Streptomyces-Ag-NPs. The biosynthesized AgNPs showed a pronounced antibacterial efficacy against Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, and Staphylococcus aureus. Moreover, the obtained Streptomyces-AgNPs exerted biofilm inhibition activity against nosocomial hospital-resistant bacteria, including Bacillus subtilis, Staphylococcus aureus, and Escherichia coli. The mechanism of biogenic AgNPs antibacterial action was visualized using TEM, which indicated the AgNPs accumulation and disruption of bacterial cell membrane function. Additionally, a molecular docking study was conducted to evaluate the binding mode of AgNPs with an Escherichia coli outer membrane. Furthermore, the cytotoxic profile of the AgNPs was evaluated toward three cell lines (MCF-7, HepG2 & HCT 116), and the low cytotoxic effects of the obtained nanoparticles indicated their possible medical application with low risks to human health.