The increasing bacterial infections and water contamination are the most critical global challenge. To address these challenges, innovative approaches are required to generate safe and effective nanoscale materials that inhibit the growth of microbes and remove hazardous contaminants from water. In this study, we described for the first time the eco-benign synthesis of acid protease mediated palladium nanoparticles (ACPs-PdNPs) using Melilotus indicus leaf extract. A series of physicochemical characterizations were applied to evaluate the size, shape and crystalline structure of ACPs-PdNPs. The surface Plasmon resonance (SPR) peak at 343 nm examined in the U.V spectra and the FTIR peaks at 591 cm¯1 confirmed the formation of as prepared NPs. Moreover, the ACPs-PdNPs possessed an extremely small particle size (5–30 nm), brown in color with spherical morphology. The as-synthesized nanoparticles were screened for their biological activities and photocatalytic applications. The catalytic efficiency of ACPs-PdNPs were inquired by the photodegradation of Rhodamine B (Rh. B) dye. Based on the findings, only 20 min of irradiation were enough to degrade 99 % of Rh. B. The ACPs-PdNPs demonstrated exceptional antibacterial activity against Escherichia coli (E. coli) bacteria in both light and dark environments, with inhibition zones of 33 mm and 27 mm, respectively. In addition, 1,1-diphenyl-2-picryl-hydrazil (DPPH) radical scavenger potential of these nanoparticles was quite strong by scavenging 88 % of the DPPH radicals. Furthermore, the Michigan Cancer Foundation (MCF-7) women's breast cancer cell lines were strongly inhibited by the acid protease mediated ACPs-PdNPs with the IC50 value of 66.37 µg/mL. Due to their trivial size and substantial surface area, ACPs-PdNPs exhibited exceptional photocatalytic and biomedical applications.