The delayed and compromised diabetic foot ulcers (DFUs) healing due to methicillin resistance staphylococcus aureus (MRSA) is an emerging threat to human health and life. Developing new strategies to clear bacteria from ulcerative regions can be helpful to treat DFUs. The current study was designed to fabricate poly (3-hydroxy butyric acid-co-3-hydroxy valeric acid, (PHBV)), nanofibers (PHBV NFs) loaded with cephalexin (CPL) by electrospining technique to improve the bioavailability of CPL at the wound site to inhibit the bacterial growth and trigger the ulcer healing process. Morphological analysis shown that the electrospun nanofibers exhibited smooth surface morphology with an average diameter ranges from 205 ± 25 to 450 ± 55 nm, depending on the cephalexin concentration. The prepared nanofibers showed an enhanced drug loading capacity (LC) and entrapment efficiency (EE) of about 12.1 ± 1.6% and 75.3 ± 2%, respectively. The successful drug loading was also confirmed by FTIR analysis. Moreover, the CPL-loaded PHBV NFs exhibited high water uptake and biodegradation rate compared to blank PHBV NFs. The drug-loaded NFs initially showed a burst drug release followed by a sustained drug release profile with a constant rate up to 48 h. In-vitro studies show that CPL-loaded PHBV NFs exhibited an enhanced antibacterial activity against MRSA strains and have no visible toxic effect against keratinocytes, L929 and HEK293T cell lines. Further, CPL-loaded PHBV NFs showed the enhanced in-vivo bacterial clearance from DFUs using NcZ10 diabetic mice model compared to free CPL. Collectively, our results demonstrated that CPL-loaded PHBV NFs can be applied as a favorable dressing material to treat MRSA-infected chronic DFUs.