Abstract Background Researchers, prompted by the toxicity and side effects associated with cisplatin, are exploring alternative approaches for developing transition metal-based anticancer agents. Employing a green biochemical approach, we transformed Nickel pyridine dicarboxylic acid compounds into the nanoscale using the aqueous extract of Macrotyloma uniflorum (horse gram). Results Characterization of the biosynthesized nanoparticles involved electronic and IR spectroscopy. A scanning electron microscope revealed a predominant spherical shape for most Nickel nanoparticles (Ni-NPs), with XRD patterns indicating particle sizes ranging from approximately 30–150 nm. The nanoparticles were evaluated for their free radical scavenging efficiency and in vitro anti-malignant properties against HeLa and A549 cancer cell lines. Numerical optimization of the DPPH and MTT assays was conducted using response surface methodology (RSM), focusing on the effects of 3,4-pyridine dicarboxylic acid (ML1), 2,4-pyridine dicarboxylic acid (ML2), nickel nanoparticles concentration, and temperature. In this investigation, the incorporation of Horse Gram seed extract (Macrotyloma uniflorum) has unveiled its abundance in phenolic and flavonoid compounds, widely acknowledged for their robust antioxidant activity in the existing literature. Conclusion The present study highlights the potential for refining the bio-toxicity and biochemical attributes of Ni-NPs to pave the way for a new generation of versatile anticancer agents with clinically established efficacy. Notably, the anticipated data closely corresponds with experimental outcomes, reinforcing the trustworthiness and validity of the RSM model for examining anticancer and antioxidant properties in this context. ML2 exhibited heightened antioxidant and anticancer activities in comparison to ML1 nanoparticles. Graphical abstract