In this study, biogenically synthesized AuNPs were first characterized via UV visible spectroscopy, SEM, XRD, and FTIR followed by toxicity evaluation using mice model. UV-visible spectroscopy of biogenic AuNPs showed peaks at 540-549 nm, while FTIR spectrum showed various functional groups involving O-H, Amide I, Amide II, O-H, C-H groups, and so on. SEM showed the size variation from 30 to 60 nm. Antibacterial potential against pathogenic isolates showed bigger ZOI (31.0 mm) against Pseudomonas aeruginosa AuNPs. Antibiofilm activity showing up to 100% inhibition at 90 µg mL-1 concentration of AuNPs. Toxicity evaluation showed LD50 as 70 mg kg-1. Exposure to AuNPs caused significant changes in the levels of serum AST (p < 0.05) at 100-150 mg kg-1 of AuNPs exposure. Histopathology of male albino mice kidney and liver revealed that mice exposed to maximum concentration of AuNPs showed necrosis, cell distortion, and hepatocytes detachment. Present study showed that biologically synthesized AuNPs possess effective antimicrobial and biofilm inhibitory potential. AuNPs strong bactericidal effect even at lower concentration suggest that NPs could have excellent potential for combating pathogens. In conclusion, nanotechnology may revolutionize human life and medical industry by developing innovative drugs with the potential to treat diseases in shorter and noninvasive time period. Hence, in vitro biosafety and experimental observations followed by in vivo outcomes are crucial in shifting the novel therapeutics into medical practice thus leading further into their future development.