The effectiveness of antibiotics toward resistant strains of bacteria has brought serious concerns related to human and animal health. Controlled drug release systems, especially those based on polymer and polymer-based nanostructures appears as a remarkable approach, once they can potentially improve the therapeutic outcomes toward bacterial infections, while requiring lower amounts of drugs. The current study was designed to investigate the incorporation and release profile of a drug loaded in biodegradable electrospun nanofibrous membranes, based on the drug-polymer interactions, as well as its ability to inhibit bacterial growth. For that purpose, nanofibrous membranes of Ecovio (R) (EC), a polymer blend composed by poly (lactic acid) (PLA)/poly (butylene adipate-co-terephthalate) (PBAT), loaded with different cloxacillin (CLOX) contents were successfully produced via electrospinning technique. Electrospun nanofibers of EC unloaded and loaded with drug presented smooth surface with a mean diameter close to 600 nm. The physical-chemical characterizations by Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) confirmed the successful drug encapsulation achieved by electrospinning technique. In vitro studies revealed that the developed drug-loaded nanofibrous membrane was successful in inhibiting S. aureus growth. The cumulative release of drug from EC nanofibrous membranes containing 20% of CLOX was demonstrated to be pH dependent, where the antibiotic release rate was much faster for pH 7.3 than that for pH 5.5. In this way, the mechanism involved in the release could be either Fickian or non-Fickian depending on the pH environment. The simple and efficient strategy presented here to develop antimicrobial nanofibrous membrane make them promising for drug delivery carrier and wound dressing applications.