Electrospinning is a desired method to produce interconnected flexible nanofibrous structures suitable for tissue engineering, drug delivery, and wound healing. Ampicillin-loaded polyurethane (PU) nanofiber mats were electrospun with the antibiotic dispersed in well-oriented nanofibers. The identification of functional groups, molecular interactions and surface morphology of the fibers were analyzed using Infrared, Raman, H-1 NMR, SEM, and TEM. Weak interactions exist between the functional groups of ampicillin and PU in electrospun fibers at ratios 1:10, 1.5:10, and 2:10. The effect of -lactam antibiotic ampicillin on the characteristics of electrospun PU was studied using XRD, TGA, and DSC. Their antibacterial property is proved by good zone of inhibition against Staphylococcus aureus and Klebsiella pneumonia. Cytotoxicity tests on the electrospun scaffolds were performed with normal human keratinocyte cells (HaCaT cells). Results indicate that ampicillin-incorporated PU scaffolds are well suited for applications in wound healing and infection control. POLYM. ENG. SCI., 55:541-548, 2015. (c) 2014 Society of Plastics Engineers