Membrane-based scaffold for bone regeneration is vastly being explored to address issues that persist in defective bone regeneration, associated with infection and inflammation. This study focused on incorporating estradiol (E2) into biodegradable polycaprolactone (PCL) electrospun nanofibrous membrane, followed by the immobilization with antibacterial chlorhexidine (CHX) through the aid of a polydopamine (PDA) grafting layer. Several analyses including field emission scanning electron microscopy (FESEM), Fourier transform infrared spectroscopy (FTIR), wettability, biodegradation, drug release, antibacterial, and cytotoxicity analyses were conducted to study the physicochemical and biological properties of the membranes. The nanofibers were constructed with an average diameter of 1.32-1.33 μm and a porosity of 51%-53%, which is accommodating bone regeneration. The grafting of PDA was not only able to improve the surface wettability, which in turn allowed controllable degradability and sustained the release of E2 and CHX from the nanofibrous membranes. The immobilization of CHX on the PCL/E2 nanofibers has greatly retarded Gram-negative Escherichia coli compared to Gram-positive Staphylococcus aureus. The in vitro cytotoxicity assay statistically depicted the ability of the fabricated nanofibrous membranes to support cell proliferation without cytotoxic effects at the cell viability above 70%. These cumulative results indicate the potential development of CHX-immobilized PCL/E2 membrane as an alternative strategy to resolve bone regeneration issues.