Clean and eco-friendly synthesis of silver nanoparticles (AgNPs) proved to have promising characteristics with impressive biomedical related potentials. Here, we have employed the green process for the synthesis of AgNPs using kenaf seed (KS) extract as a bilateral mediator of reducing and capping of Ag+ ions under hydrothermal condition. The synthesis pathways, such as varying amounts of KS, silver ions concentration, and autoclaving time were optimized. A manifestation of a strong absorption peak from 420 to 430 nm in UV-Vis spectroscopy indicated fruitful synthesis of [email protected] Fourier transform infrared spectroscopy confirmed the presence of hydroxyl and carbonyl functionalities to be involved in the reduction and stabilization of Ag+ ions. Besides, Transmission electron microscopy revealed that the [email protected] possess 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 of Ag+ ions was released from [email protected] In cell line studies, [email protected] at a higher dose was unveiled as 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 [email protected] with ampicillin exposed 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 their original feature as an anti-cancer and antimicrobial agent. © 2020 IOP Publishing Ltd.