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In this study, biocompatible electrospun nanofiber scaffolds were produced using poly(-caprolactone (PCL)/chitosan (CS) and Nigella sativa (NS) seed extract, and their potential for biomedical applications was investigated. Scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), total porosity measurements, and water contact angle measurements were used to evaluate the electrospun nanofibrous mats. Additionally, the antibacterial activities of Escherichia coli and Staphylococcus aureus were investigated, as well as cell cytotoxicity and antioxidant activity, using MTT and DPPH assays, respectively. The obtained PCL/CS/NS nanofiber mat was observed by SEM to have a homogeneous and bead-free morphology, with average diameters of 81.19 ± 4.38 nm. Contact angle measurements showed that the wettability of the electrospun PCL/Cs fiber mats decreased with the incorporation of NS when compared to the PCL/CS nanofiber mats. Efficient antibacterial activity against S. aureus and E. coli was displayed, and an in vitro cytotoxic assay demonstrated that the normal murine fibroblast cell line (L929 cells) remained viable after 24, 48, and 72 h following direct contact with the produced electrospun fiber mats. The results suggest that the PCL/CS/NS hydrophilic structure and the densely interconnected porous design are biocompatible materials, with the potential to treat and prevent microbial wound infections. [ABSTRACT FROM AUTHOR]