This study investigates the green synthesis of silver nanoparticles (PF-AgNPs) using the aqueous extract of the medicinal plant Passiflora foetida to enhance their biological activity. PF-AgNPs were characterised using UV-Vis spectroscopy, FTIR, XRD, SEM, and EDX analyses. The UV-Vis absorption peaks of PF-AgNPs were observed between 380 and 480 nm. Structural characterisation using FTIR and XRD confirmed that the functional groups of P. foetida metabolites acted as surface-capping agents, yielding crystallised AgNPs with a face-centred cubic (fcc) structure. SEM-EDX analysis further revealed that the synthesised PF-AgNPs were spherical and displayed a uniform distribution of silver at 66.39% under the optimised condition. The synthesis of PF-AgNPs was optimised using a Box–Behnken design via Response Surface Methodology (RSM), evaluating the effects of extract concentration (0.1%, 0.2%, and 0.3%), pH (4, 7, and 10), and temperature (30 °C, 50 °C, and 70 °C), with observations taken at 1 and 24 hours. UV-Vis intensity, NO scavenging activity, and DPPH scavenging activity were used as the response variables in RSM modelling. All models yielded p-values of less than 0.05, indicating that the factors significantly influenced PF-AgNP synthesis, with pH identified as the most effective parameter. PF-AgNPs exhibited DPPH and NO inhibitory activities of 35.64% and 38.7%, respectively, both of which were higher than those of the extract (17.16%). These findings highlight a sustainable and statistically guided approach for synthesising bio-functional AgNPs with promising potential for future biomedical applications.