This study was aimed to synthesise antimicrobial and therapeutic silver nanoparticles (AgNPs) by green synthesis method using Vitex agnus‐castus L. (VAC) seed extract. The synthesised VAC seed extract coated AgNPs were characterised by UV‐Vis spectrometry, Zeta analysis and FT‐IR spectrometry. The antifungal, antimicrobial, cytotoxicity by MTT assay, and anticarcinogenic activities on HeLa cells line were investigated. VAC‐AgNPs exhibited high antimicrobial activity against S. aureus with a zone diameter of 11.3±0.6 mm. In addition, the cytotoxicity of VAC‐AgNPs was evaluated using the L929 fibroblast cell line and the HeLa cells line, with AgNPs applied at six different concentrations. At a concentration of 5.0 mM, viability was found to be 47.6 %±4.2 % in L929 fibroblast cells and 39.6 %±7.9 % in HeLa cells, suggesting a potential antiproliferative effect of VAC‐AgNPs on HeLa cancer cells. The IC50 values of VAC‐AgNPs against the HeLa cell line were calculated as 2.94 μg/ml.