Two series of polyurethane (PU), based on polycaprolactone (PCL) as soft segments with two different molecular weights (2000 and 530 Da), and hexamethylene diisocyanate (HDI) and 1,4-butandiol (BDO) as hard segments were synthesized to fabricate curcumin-loaded electrospun nanofibrous PCL-based PU substrate. Chemical structures of the synthesized PUs were characterized by FTIR and NMR spectroscopy techniques. The thermal properties were analyzed by differential scanning calorimetry (DSC) and surface hydrophilicity was studied by static contact angle and bulk hydrophilicity was evaluated by water uptake test. Thereafter, bead-free PU nanofiberous substrate containing curcumin was fabricated by electrospinning and morphology of the mats was observed by scanning electron microscopy (SEM). Mechanical properties of the electrospun mats in comparison with polymeric films were assessed by a universal test machine. The in vitro release of curcumin was studied by UV-Vis spectroscopy. The optical density of the bacterial solutions was used to evaluate the antibacterial activity of the curcumin-loaded nanofibrous mats against Escherichia coli (E-coli ATCC: 25922). The results showed that curcumin-loaded PU synthesized by PCL with molecular weight of 2000 Da displayed better mechanical properties as well as better antibacterial properties in wound dressing application.