The demand for advanced, biocompatible wound dressings with antibacterial properties is increasing in order to treat people with severe skin wounds, such as burn victims or those suffering from ulcers. We have developed an ultrafine three-layer polymer nanofiber mesh using electrospinning that is able to kill bacteria (Escherichia coli; E. coli and Staphylococcus aureus; S. aureus) but also has cytocompatibility properties. The first layer was generated with polystyrene (PS) for strength and functions as a carrier layer. The second layer consisted of polycaprolactone (PCL) with silver nanoparticles (Ag NPs) that were added to the spinning solution, which had antibacterial properties. Finally, the third layer comprised of polyethylene oxide (PEO) acting as a hydrophilic, barrier layer that was also non-adhesive, with the potential to further assist in wound healing. Systematic physicochemical and biological characterization was performed including dynamic light scattering (DLS), UV spectroscopy, X-ray photoelectron spectroscopy (XPS), X-ray diffraction (XRD), inductively coupled plasma optical emission spectroscopy (ICP-OES), scanning electron microscopy (SEM), water contact angles, evaluation of antibacterial properties, and cell attachment and proliferation assays. The cumulative Ag ion release was optimized for a period up to 84 days in physiologically similar media at physiological temperature. Chemical, mechanical, and biological analysis demonstrated that incorporation of Ag NPs at higher quantities into the PCL fibers layer providing excellent antimicrobial activity with minimal toxicity at low concentration. The findings highlight the importance of optimizing the properties of Ag based antibacterial meshes to find the balance between high antibacterial activity and low toxicity. © 2022 Elsevier B.V.