This work reports the cytotoxic and antibacterial activities of starch encapsulated phytogenic silver nanoparticles synthesized from Paeonia lactiflora flowers (PLF-AgNPs). To optimize the synthesis of AgNPs, a total of 13 reactions were performed with the different combinations of AgNO₃ (1–5 mM), plant extract (5–15% v/v), and light exposure time (0–48 h). The UV-spectrum results indicated that AgNPs were not produced without the light exposure while the light exposure significantly triggered the production of AgNPs. Among the various combination of plant extracts, AgNO₃ concentration, and light exposure, only four combinations were successfully formed the AgNPs and they were named as PLF-AgNPs (1:5:48), PLF-AgNPs (3:5:24), PLF-AgNPs (3:15:3.5), and PLF-AgNPs (3:5:3) according to their ratio of AgNO₃:plant extract:light exposure time. The physicochemical properties of newly synthesized all the NPs were studied using the various characterization techniques including FTIR, XRD, and FE-TEM, EDS. The encapsulation of the starch was significantly reduced the toxicity of AgNPs in the NIH3T3 cell line and LN229 cell line evidenced by cytotoxicity, AO/EB, and DACF-DA staining assay. Besides, ST-PLF-AgNPs has inhibited the bacterial growth higher than the PLF-AgNPs with MIC of 3.91 ± 0.11, 1.63 ± 0.09, 2.86 ± 0.39, 1.95 ± 0.24 μg/mL for Listeria monocytogenes, Staphylococcus aureus, Escherichia coli, and Salmonella enterica, respectively. The present results proved that encapsulation of the starch reduced the cytotoxicity by increasing the antibacterial activity of ST-PLF-AgNPs, which is deserved for biotherapeutics analysis to treat bacterial infections.