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Rubia cordifolia L. is a widely used traditional medicine in the Indian sub-continent and Eastern Asia. In the present study, the aqueous leaf extract of the R. Cordifolia was used to fabricate silver nanoparticles (RC@AgNPs), following a green synthesis approach. Effect of temperature (60 °C), pH (8), as well the concentration of leaf extract (2 ml) and silver nitrate (2 mM) were optimized for the synthesis of stable RC@AgNPs. The phytofabrication of nanosilver was validated by UV–visible spectral analysis, which displayed a distinctive surface plasmon resonance peak at 432 nm. The effective functional molecules as capping and stabilizing agents, and responsible for the conversion of Ag+ to nanosilver (Ag0) were identified using the FTIR spectra. The spherical RC@AgNPs with an average size of ~ 20.98 nm, crystalline nature, and 61% elemental composition were revealed by TEM, SEM, XRD, and. EDX. Biogenic RC@AgNPs displayed a remarkable anticancer activity against B16F10 (melanoma) and A431 (carcinoma) cell lines with respective IC50 of 36.63 and 54.09 µg/mL, respectively. Besides, RC@AgNPs showed strong antifungal activity against aflatoxigenic Aspergillus flavus, DNA-binding properties, and DPPH and ABTS free radical inhibition. The presented research provides a potential therapeutic agent to be utilized in various biomedical applications. [ABSTRACT FROM AUTHOR]