One of the popular subjects of millennia is the synthesis of nanostructures, their applications in numerous fields, and their interaction with various biological systems, making them appealing for drug delivery systems, and diagnostic and therapeutic agents. In this study, silver oxide nanoparticles were synthesized using E. sativa (ES) aqueous extract. The biosynthesis was followed via UV-vis spectroscopy by analysis, FTIR, XRD, TEM, and Zeta potential to further analyze the synthesized nanoparticles. Furthermore, the biosynthesized silver oxide nanoparticles (AgONPs) were checked through various biological activities. The antioxidative potential was assessed by performing a DPPH radical scavenging assay, total reducing power assay, and total antioxidant capacity assay. Antimicrobial potential was observed against various bacterial and fungal strains. Likewise, Artemia salina (brine shrimps) was used to study cytotoxicity, while VERO and HEK-293 cell lines were applied to check the biocompatibility of synthesized NPs. Anticancer potential was evaluated against the Hep-2 cells by utilizing an MTT assay. A mean crystallite ~ 50 nm size is evidenced by TEM analysis. Notable antimicrobial activity was detected with various bacterial and fungal strains with maximum ZOI by B. subtilis was 18.5 ± 2.36 mm at 1000 µg/mL and A. niger reveals a minimum ZOP of 16 ± 1.7 mm at 1000 µg/mL respectively. A dose-dependent response was observed in biological evaluation against A. salina (LC: 12.21 µg/mL), DPPH (IC: 62.36 µg/mL), VERO cell line (IC: 43.11 µg/mL), HEK-293 cell line (IC: 26.56 µg/mL), and Hep-2 cell line (IC: 9.97 µg/mL). The multifaceted attributes of ES-AgONPs encompassing antimicrobial, antioxidant, cytotoxic, and anticancer properties render them a versatile platform in drug delivery and biomedical horizons. However, detailed investigation and clinical trials will undoubtedly provide translational applications in diverse fields.