The clean and eco-friendly synthesis of silver nanoparticles (AgNPs) has provided promising characteristics with impressive biomedical related potential. Here, we have employed a green process for the synthesis of AgNPs using kenaf seed (KS) extract as a bilateral mediator for reducing and capping of Ag⁺ ions under hydrothermal condition. The synthesis pathways, such as varying amounts of KS, Ag ion concentration and autoclaving time were optimized. The manifestation of a strong absorption peak from 420–430 nm in UV–vis spectroscopy indicated the successful synthesis of KS@AgNPs. Fourier transform infrared spectroscopy confirmed the presence of hydroxyl and carbonyl functionalities involved in the reduction and stabilization of Ag⁺ ions. Furthermore, transmission electron microscopy revealed that the KS@AgNPs are spherical in shape having a size around 7–11 nm, whereas high-quality crystals were evidenced by x-ray diffraction analysis. Moreover, inductively coupled plasma-optical emission spectrometry revealed that 19.6 μg l⁻¹ of Ag⁺ ions were released from the KS@AgNPs. In cell line studies, KS@AgNPs at a higher dose were shown to be non-toxic to the healthy (NIH3T3) cells, while strong anti-proliferative response was found in the case of lung cancer (A549) cells. Furthermore, a significant zone of inhibition was observed for both Gram-positive and Gram-negative microorganisms, and a combination of KS@AgNPs with ampicillin revealed a notable synergistic anti-pathogenic effect. Overall, our study proved the potentiality of KS as an efficient bio-resource for the synthesis of AgNPs and also its original feature as an anti-cancer and antimicrobial agent.