Green synthesis of silver nanoparticles (AgNPs) has gained attention as an eco-friendly and sustainable approach to nanomaterial production, particularly in the search for alternatives to conventional antimicrobials amid rising resistance. This systematic review, conducted in accordance with PRISMA 2020 guidelines, identified 17 in vitro experimental studies that investigated the antimicrobial potential of green-synthesized AgNPs. Biological sources included plants (n = 11), fungi (n = 2), a polysaccharide (n = 1), a cyanobacterium (n = 1), and a succulent (n = 1). Reported nanoparticle sizes ranged from 8 to 150 nm, with smaller particles (<30 nm) generally exhibiting superior antimicrobial efficacy. Antimicrobial activity was demonstrated against Gram-positive bacteria in 15 studies, Gram-negative bacteria in 14 studies, and fungi in 5 studies, with zones of inhibition ranging from 7 mm to 37 mm. Only six studies reported minimum inhibitory or bactericidal concentrations, underscoring a lack of standardized quantitative data. The predominant mechanisms of action were attributed to reactive oxygen species (ROS) generation, oxidative stress, membrane disruption, protein inactivation, and DNA interference. Cytotoxicity was assessed in six studies, suggesting biocompatibility at lower concentrations but potential dose-dependent toxicity. Overall, green-synthesized AgNPs demonstrate consistent antimicrobial potential, but future research must focus on standardized synthesis protocols, robust MIC/MBC testing, and systematic toxicity evaluation to support clinical translation.