Silver nanoparticles (AgNPs) have been recognized for their outstanding antibacterial activities, which are required for antibacterial coating materials in therapeutic applications. A bacterialresistant electrospun nanofibrous mat made of polycaprolactone (PCL) in combination with polylactide acid (PLA) containing silver nanoparticles encapsulated with Thymus vulgaris L. (thyme) extract (eAgNPs) was fabricated in order to assess the potential of applicability in biomedical applications such as cancer treatment, wound healing, or surgical sutures. In the current study, PCL and PLA used as the basis polymers were blended with biosynthesized eAgNPs, pure AgNPs, and thyme extract (TE) to observe the effects of additives in terms of antibacterial and anticancer activity and morphologic, thermal, mechanical, biocompatibility, and biodegradability properties. The biological characteristics of fabricated electrospun nanofibrous mats were evaluated in vitro. Physicochemical characteristics of the nanofibrous mats were examined by UV−vis spectrophotometry, scanning electron microscopy (FE-SEM), transmission electron microscopy (TEM), energy dispersive X-ray (EDX), Fourier-transform infrared spectroscopy (FTIR), mechanical tensile testing, X-ray diffraction (XRD), thermogravimetric examination (TGA), and water contact angles (WCAs). The results showed that a biodegradable nanofiber scaffold with a mean fiber diameter of 280 nm is morphologically homogeneous and highly hydrophobic, has higher tensile strength than PCL/PLA nanocomposite fiber, and is resistant to Escherichia coli and Staphylococcus aureus. The cytotoxic and anticancer properties of nanomaterials were defined using L929 and SK-MEL-30 cells. The developed material inhibited cell proliferation and led to apoptosis of cell lines. It can be suggested that the use of Thymus vulgaris L. extract-encapsulated silver nanoparticle-doped PCL/PLA nanofibers produced by the electrospinning method has the potential for cancer therapy in skin tumor cell lines.