The current study aimed to formulate zinc oxide nanoparticles (ZnONPs) employing an aqueous extract derived from Rhynchosia capitata (RC). Utilizing several spectroscopic methods (UV-visible spectroscopy, XRD, FTIR, SEM, EDX, DLS), the synthesis of ZnONPs was evaluated. ZnONPs have a mean crystallite size of about 32.15 nm, as determined by the Scherrer equation. Moreover, in vitro, biological assessments showed notable therapeutic potential. Furthermore, the disc diffusion method was precisely employed with antibacterial and antifungal properties across a range of concentrations (100–1500 µg/ml). Among the bacteria, B. subtilis displayed a prominent inhibition zone, measuring 24 mm at 1500 µg/ml. Similarly, susceptible fungal strains demonstrated a zone of inhibition measuring 29 mm. Antioxidant potentials were determined using different assays. The radical-scavenging capabilities were quantified using a DPPH assay and revealed a significant result of 73%. Further, reducing power was calculated and found to be 69 ± 1.77%, and the total antioxidant capacity was found to be 88 ± 4.8. Brine shrimps cytotoxicity assay was performed and displayed an LC50 value of 2.20 µg/mL, affirming biocompatibility. The biocompatibility potential of ZnONPs was determined against red blood cells, HEK, and VERO cell lines at different concentrations to confirm their biosafety and biocompatibility. An alpha-amylase (AA) inhibition assay was performed and showed a percent inhibition of 70%. Particularly noteworthy was the ZnONPs efficacy against (Hep-2 liver) cancer cells (IC50:44.90). The ZnONPs exhibited promising non-toxic attributes and displayed substantial potential for diverse biological applications in various domains. The outcomes of this study are anticipated to contribute valuable insights and open new avenues for further research and development of ZnONPs in a multitude of biomedical applications.