A multilayered polylactic acid (PLA)/halloysite (HAL) porous membrane encapsulated with aminoglycoside antibiotic (gentamicin) was prepared by electrospinning technique. The prepared electrospun membrane displayed smooth morphology and uniform fiber distribution, while various layers of PLA mats were tightly bounded together. Significant enhancement in mechanical properties and stability was observed in membranes reinforced by HAL and the multilayered membrane loaded with gentamicin, while the drug loaded membrane showed no delamination. In addition, incorporation of HAL led to improvement in thermal stability of the PLA nanofibrous membranes. The in vitro drug release study of gentamicin loaded membrane initially shows a burst release of gentamicin, followed by slow rate of drug release up to 48 h. Antimicrobial efficacy of electrospun membrane was tested on a gram-positive (Staphylococcus aureus) and a gram-negative (Escherichia toll) bacteria, while results indicated that the gentamicin released from electrospun membrane has retained its antibacterial activity. These results signified the potential of multilayered porous PLA/HAL membranes to be utilized in prevention of infection in bone regeneration applications.