Metallic nanoparticles are very useful, effective, and usually synthesized by toxic and expensive chemicals. Silver nanoparticles (AgNPs), measuring less than 100 nm, have shown promising impact in several biomedical investigations. These can inhibit microbial growth and aid in medicine administration. Six substrates of Carica papaya were used to synthesize silver nanoparticles that can limit the growth of bacteria and fungi. In this article, we report the synthesis of AgNPs from the leaf, seed, callus, peel, fruit juice, and bark of Carica papaya. AgNPs synthesized from callus showed the most promising results when tested against the growth of bacteria like Xanthomonas campestris, Erwinia carotovera, Bacillus subtilis, and fungi (Aspergillus niger and Fusarium oxysporum) when compared with other extracts’ efficacy, and the callus was regenerated from petiole and midrib explants of Carica papaya in MS basal media supplemented with NAA and Kinetin (1 + 0.5 mg/L). A ratio of 1:20 of substrate extract to 1 mM AgNO3 produced the most effective nanoparticles in terms of capping, quality, and stability when tested through surface plasmon resonance (SPR) within the 400–435 nm range. The nanoparticle sizes of all six types were measured using Image J software on micrographs of SEM at 200 nm resolution. The average diameters were analyzed through Origin software, and the finest AgNPs were observed to be synthesized from callus extract, i.e., 18.91 nm with rod-like morphology. Energy dispersive X-ray (EDX) at 2.6 keV revealed 43.38, 75.39, 70.611, 36.54, 58.57, and 45.94 percent elemental silver in AgNPs formed from the leaf, callus, juice, seed, bark, and peel extract, respectively. Silver nanoparticles synthesized from callus extract were smaller and exhibited the most effective antimicrobial potential, with the highest inhibitory zone of 19 mm against Xanthomonas campestris bacterium and up to 14 mm against Aspergillus niger fungus. Furthermore, the percentage of elemental Ag (measured through EDX) was found to be highest in the nanoparticles synthesized from callus compared to those synthesized from the leaf, seed, peel, fruit juice, and bark of Carica papaya. Hence, the callus extract is the most suitable substrate for the reduction of silver nitrate solution in 1:20 to form the finest silver nanoparticles in an effective biogenic way.