The synthesis of well-dispersed and ultrafine metal nanoparticles has great interest due to their distinctive physicochemical properties and biomedical applications. This study is the first report of one-step solvent-free synthesis of AgNPs using Euphorbiaceae plant latex. Among evaluated eight latex-producing plants, four (Jatropha curcasJatropha gossypifoliaPedilanthus tithymaloides, andEuphorbia milii) showed high potential to produce physicochemically distinct, small-sized and bactericidal AgNPs. Phytochemical screening showed presence of rich amount of biochemicals in these plants. J. gossypifolia showed uniformly dispersed comparatively small-sized AgNPs. Dose-dependent growth inhibition of bacterial pathogens Escherichia coliPseudomonas aeruginosa,Staphylococcus aureusStaphylococcus epidermis, and Micrococcus luteus was observed for J. gossypifolia latex-synthesized AgNPs with minimum inhibitory concentration values 30, 40, 70, 60, and 60 ppm, respectively, after 24 h. Possible mode of action of AgNPs against pathogens was confirmed by analyzing enzymes and cell leakage.