Nanoparticles have gained worldwide attention as a new alternative to chemical control agents due to their special physiochemical properties. The current study focused on the environmentally friendly synthesis of silver nanoparticles (AgNPs) using Lycopersicon esculentum peel. In addition to studying the intrinsic cytotoxic effectiveness of Le-AgNPs contribute to their antibacterial, and antifungal activities and the effect of nanoparticles on the integrity of their morphological behavior. The initiative biosynthesis of L. esculentum silver nanoparticles (Le-AgNPs) was indicated by the color change of L. esculentum (Le) extract mixed with silver nitrate (AgNO3) solution from faint pink to faint brown. UV-visible spectroscopy, Dynamic light scattering (DLS), Fourier-transform infrared spectroscopy, high-resolution transmission electron microscopy (HR-TEM), and X-ray diffraction techniques were used to characterize biosynthesized Le-AgNPs. Results of UV-visible spectroscopy recorded surface plasmon resonance at 310nm for SPR of 2.5. The DLS results showed particles of 186nm with a polydispersity index of 0.573. The FTIR spectrum indicated the existence of carboxyl, hydroxyl, phenolic, and amide functional groups. The HR-TEM analysis revealed quasi-spherical crystal particles of Le-AgNPs. Le-AgNPs had a negative zeta potential of - 68.44mV, indicating high stability. Bacillus subtilis ATCC 6633 and Escherichia coli ATCC 8739 were the most susceptible pathogens to Le-AgNPs inhibition, with inhibition zone diameters (IZDs) of 4.0 and 0.92cm, respectively. However, Listeria monocytogenes NC 013768 and Shigella sonnei DSM 5570 were the most resistant pathogens, with IZDs of 0.92 and 0.90cm, respectively. Le-AgNPs demonstrated good inhibitory potential against pathogenic fungi, with IZDs of 3.0 and 0.92cm against Alternaria solani ATCC 62102 and Candida albicans DSM 1386, respectively. The cytotoxicity effect was observed at a half-maximal inhibitory concentration (IC50) of 200.53μg/ml on human colon NCM460D normal cells.