Background: Gold nanoparticles (AuNPs), unlike chemotherapy, may destroy cancer cells without harming normal cells. Objective: For the first time, Acinetobacter (A.) baumannii-biosynthesized AuNPs (Ab-AuNPs) were evaluated for anticancer properties. Materials and methods: The Ab-AuNPs were characterized by UV-visible spectroscopy, TEM, FE-SEM, AFM, XRD analysis, and FTIR. The MTT assay was used to determine the antiproliferative efficacy of the Ab-AuNPs at different concentrations against prostate cancer (PC3) and human breast normal cell line (MCF-10). Potential apoptotic-mediated cell death was investigated using acridine orange and propidium iodide staining and mitochondrial ‎membrane potential (MMP). Results: Biosynthesized Ab-AuNP had a zeta potential below 30 mV at -22 mV, suggesting particles stability with no aggregation. The average size was estimated at 66 nm (range 20-90 nm). Ab-AuNPs inhibited PC3 cell proliferation dose-dependently, with no adverse effect on MCF-10 cells. The mean lethal dose (IC₅₀) was 13.72 µg/mL, and the maximal inhibitory concentration was 25.50 mg/mL (57.33% and 80.67%, respectively). Examining PC3 cancer cells treated with Ab-AuNPs at IC₅₀ for 24 hours by flow cytometry revealed an ‎increase in apoptosis that resulted in the disruption of the membrane and vacuoles of the lysosomes, which ‎corresponded with the depletion of MMP. This was in contrast to MCF-10 AuNPs-treated cells, which showed no adverse effects. Conclusions: Producing gold nanoparticles in an A. baumannii broth culture is simple, inexpensive, and non-toxic to healthy ‎cells. They showed anticancer effects by inducing apoptosis, making ‎them a ‎potential effective anticancer agent.