Silver Nanoparticles (AgNPs) are widely used in medical and pharmaceutical applications due to their antimicrobial properties. In this study, Ag-NPs were synthesized using Viola tricolor aqueous extract as a reducing and capping agent. The presence of synthesized AgNPs was first confirmed with UV-Visible, SEM, TEM, XRD, and FTIR analyses, and then their antimicrobial characteristics were studied based on the Minimum Inhibitory Concentration (MIC). The SEM analysis showed that the synthesized AgNPs were spherical in shape. The particle size histogram revealed that the average particle size of the AgNPs was 49.45 nm. Findings from the FTIR and UV-Vis spectra showed the successful formation of Ag-NPs because the functional groups involved in the synthesis process and adsorption peaks were well developed. Furthermore, the Ag-NPs had a peak absorption at 420 nm in the spectrometry. MIC results showed the strong antimicrobial effects of the synthesized Ag-NPs. Results of the Minimum Bactericidal Concentration (MBC) revealed the dosedependent cytotoxicity of the Ag-NPs. Nanoparticles could exert the inhibitory effect of DDPH free radicals in a dose-dependent manner. Methyl Tetrazolium (MTT) results showed that silver nanoparticles had a dose-dependent cytotoxic effect and significantly reduced cell survival. The IC50 values for Ag-NPs and the extract were 11.83 and 204.4 μg mL-1 , respectively. This study showed a higher cytotoxic effect of the green synthesized nanoparticles on hepatocellular carcinoma cells than the extract. Consequently, the biosynthesis of Ag-NPs using aqueous extract of V. tricolor is clean, inexpensive, and has antibacterial, anticancer and antioxidant activities. Hence, it can be used as a drug candidate.