This study reports the synthesis of silver nanoparticles using amino acid L-histidine as a reducing and capping agent as an eco-friendly approach. Fabricated L-histidine-capped silver nanoparticles (L-HAgNPs) were characterized by spectroscopic and microscopic studies. Spherical shaped L-HAgNPs were synthesized with a particle size of 47.43 ± 19.83 nm and zeta potential of −20.5 ± 0.95 mV. Results of the anticancer potential of L-HAgNPs showed antiproliferative effect against SiHa cells in a dose-dependent manner with an IC50 value of 18.25 ± 0.36 µg/mL. Fluorescent microscopic analysis revealed L-HAgNPs induced reactive oxygen species (ROS) mediated mitochondrial dysfunction, leading to activation of apoptotic pathway and DNA damage eventually causing cell death. To conclude, L-HAgNPs can act as promising candidates for cervical cancer therapy.