Nanofibers have attracted a lot of interest in the biomedical field, specifically in nanoscience and nanotechnology. It is challenging to use electrospinning for the achievement of novel nanofibrous composites by the use of inorganic precursors without beads and with a uniform diameter. In this research, the development of biocompatible conjugate electrospinning nanofiber membranes containing zeolite was studied. Nanofiber membranes based on polyestersulfone as the first component and polyvinyl alcohol loaded with zeolite as the second component were prepared using the conjugate electrospinning method. Smooth, uniform nanofibers without the beads containing zeolite in the polymer substrate were observed by scanning electron microscopy (SEM). Conjugate electrospinning nanofiber membranes showed good tensile strength (3/42 MPa) and elastic modulus (48/5 MPa) after thermal crosslinking. In addition, the contact angle was reduced by the addition of polyvinyl alcohol/zeolite, which could eliminate the limitations of polyethersulfone-based membranes due to their high hydrophobicity. The MTT results and cell culture also showed that the polyester sulfone/polyvinyl alcohol-zeolite conjugate electrospinning nanofiber scaffolds made in this study have excellent biological and non-toxic compatibility for the growth of L929 fibroblasts. Therefore, PES/PVA-zeolite conjugate electrospinning nanofiber membranes can provide biomedical applications.