This study describes a simple, cost-effective, and eco-friendly method for synthesizing silver nanoparticles using a rosmarinic acid extract from Perilla frutescens (PFRAE) as the bioreduction agent. The resulting nanoparticles, called PFRAE-AgNPs, were characterized using various analytical techniques. The UV-Vis spectrum confirmed the formation of PFRAE-AgNPs, and the FTIR spectrum indicated the participation of rosmarinic acid in their synthesis and stabilization. The XRD pattern revealed the crystal structure of PFRAE-AgNPs, and the TEM analysis showed their spherical morphology with sizes ranging between 20 and 80 nm. The DLS analysis indicated that PFRAE-AgNPs were monodispersed with an average diameter of 44.0 ± 3.2 nm, and the high negative zeta potential (-19.65 mV) indicated their high stability. In the antibacterial assays, the PFRAE-AgNPs showed potent activity against both Gram-positive (Bacillus subtilis and Staphylococcus aureus) and Gram-negative (Escherichia coli and Pseudomonas aeruginosa) bacterial pathogens, suggesting that they could be used as a potential antibacterial agent in the clinical setting. Moreover, the antioxidant activity of PFRAE-AgNPs against DPPH and ABTS radical scavengers highlights their potential in the treatment of various oxidative stress-related diseases. PFRAE-AgNPs also demonstrated significant anticancer activity against a range of cell lines including human colon cancer (COLO205), human prostate carcinoma (PC-3), human lung adenocarcinoma (A549), and human ovarian cancer (SKOV3) cell lines suggesting their potential in cancer therapy. The nanoparticles may also have potential in drug delivery, as their small size and high stability could enable them to cross biological barriers and deliver drugs to specific target sites. In addition to the aforementioned properties, PFRAE-AgNPs were found to be biocompatible towards normal (CHO) cells, which is a crucial characteristic for their application in cancer therapy and drug delivery systems. Their antibacterial, antioxidant, and anticancer properties make them promising candidates for the development of new therapeutic agents. Furthermore, their small size, high stability, and biocompatibility could enable them to be used in drug delivery systems to enhance drug efficacy and reduce side effects.