Electrospun polymeric nanofibers have numerous attractive biomedical applications owing to their structural resemblance with the natural extracellular matrix as well as their possibility of being loaded with different bioactive agents. The present work aimed to investigate the antibacterial activity and wound healing potential of biodegradable electrospun PVA/MMT nanofibers containing root extract of Berberis lycium. The study involved the preparation and characterization of PVA/MMT nanofibers incorporated with root extract of Berberis lycium. The antibacterial activities of fabricated nanofiber were performed against two bacterial strains, Staphylococcus aureus and Pseudomonas aeruginosa, by agar diffusion method. The extract loaded and unloaded nanofibers were subjected to the in vivo wound healing studies using rabbits and compared with standard and control groups. The antibacterial potential of electrospun nanofibers was greatly influenced by the concentration of root extract, i.e. a direct relation between the antibacterial activity and extract concentration. The highest percentage of wound closure was shown by the test group as compared with the control group, showing the good healing potential of extract-loaded nanofibers. The PVA/MMT nanofibers incorporated with root extract of Berberis lycium possess promising antibacterial activity and healing potential against burn wounds.