Although guided tissue regeneration (GTR) is a useful tool for regenerating lost tissue as bone and periodontal tissue, a biocompatible membrane capable of regenerating large defects has yet to be discovered. This study aimed to characterize the physicochemical properties and biological compatibility of polycaprolactone (PCL) membranes associated with or without nanostructured hydroxyapatite (HA) (PCL/HA) and Zn-doped HA (PCL/ZnHA), produced by electrospinning. PCL, PCL/HA, and PCL/ZnHA were characterized by field emission scanning electron microscopy (FESEM), transmission electron microscopy (TEM), energy dispersive X-ray spectroscopy (EDS), X-ray diffraction (XRD), thermal gravimetric analysis (TGA), and differential scanning calorimetry (DSC). Nanoparticles of HA or ZnHA were homogeneously distributed and dispersed inside the PCL fibers, which decreased the fiber thickness. At 1 wt% of HA or ZnHA, these nanoparticles acted as nucleating agents. Moreover, HA and ZnHA increased the onset of the degradation temperature and thermal stability of the electrospun membrane. All tested membranes showed no cytotoxicity and allowed murine pre-osteoblast adhesion and spreading; however, higher concentrations of PCL/ZnHA showed less cells and an irregular cell morphology compared to PCL and PCL/HA. This article presents a cytocompatible, electrospun, nanocomposite membrane with a novel morphology and physicochemical properties that make it eligible as a scaffold for GTR.