Abstract In this study, Cannabis sativa roots extract has been employed for the biosynthesis of silver nanoparticles (AgNPs). The appearance of reddish-brown colour followed by absorption peak of AgNPs at 408 nm through UV-vis spectrophotometry suggested biosynthesis of AgNPs. The size of the particles ranged from 90–113 nm, confirmed using DLS and TEM along with zeta potential of −25.3 mV. The FTIR provided information regarding the phytochemical capping. The study was further elaborated for determining AgNPs antibacterial, antioxidant, and cellular toxicity using MIC, DPPH, MTT, and haemolytic assays, respectively. The AgNPs were significantly effective against Staphylococcus aureus (Gram-positive), as compared to that of Pseudomonas aeruginosa, Klebsiella pneumoniae,...