Wound infections caused by multidrug-resistant bacteria present a substantial challenge in healthcare. Nanofibers, particularly when infused with natural extracts, are emerging as promising platforms for antimicrobial applications. This study investigates the potential of Anastatica hierochuntica extract-loaded electrospun nanofibers prepared with thermoplastic polyurethane for combating infections and promoting wound healing. Electrospinning was utilized to prepare nanofibers infused with Anastatica hierochuntica extract, resulting in uniform rod-shaped structures confirmed by scanning electron microscopy. The hydrophilicity of the nanofibers was assessed through water contact angle (WCA) measurements and swelling tests. Mechanical properties, including strain and stress were evaluated to determine suitability for drug delivery. The formulation with optimal properties, designated as NF20, underwent further investigation. Drug release profiles were analyzed over 72 h, and antimicrobial efficacy was tested against various pathogens, with comparisons made to Silymarin as a standard. A biofilm study evaluated the anti-virulence activity, while wound healing assays assessed the optimized extract loaded nanofibers potential in fostering tissue repair. The extract-loaded nanofibers exhibited enhanced hydrophilicity, with a WCA of 43.1 ± 0.6° and swelling of 216.67 ± 2.36% after 1 h. NF20 demonstrated superior mechanical properties, with strain and stress values of 67.6% and 0.0486 N/mm, respectively. The sustained release profile indicated 73.40 ± 1.31% release after 72 h. Antimicrobial tests revealed significant reductions in minimum inhibitory concentration, minimum bactericidal concentration, and minimum fungicidal concentration against key pathogens. The biofilm study confirmed extract loaded nanofiber's efficacy in inhibiting biofilm formation and disrupting established biofilms. These findings underscore the potential of the extract-loaded nanofiber composed of thermoplastic polyurethane as innovative wound dressings that enhance antimicrobial properties, promote accelerated healing and support tissue regeneration.