Chitosan-based nanofibers have become attractive biomaterials for wound healing and dressing applications based on their intrinsic biocompatibility, biodegradability, and antibacterial properties. However, the unstable spinnability of chitosan-based nanofibers has impeded further applications. In this paper, a fatty acid amide oleamide was used as a blending material for nanofiber fabrication. The addition of oleamide into chitosan moderately decreased the viscosity of the electrospinning solution, resulting in enhanced spinnability when constructing chitosan/oleamide blended nanofibers. Remarkably, the 1 : 0.5 ratio of chitosan/oleamide nanofibers exhibited relatively high hydrophobicity, decreased tensile strength, and increased elongation at break compared to chitosan-only nanofiber. The nanofiber showed similar and slightly higher cell adhesion in the in vitro cell culture with mouse preosteoblast MC3T3-E1 and fibroblast NIH/3T3 cells, respectively; however, the cell proliferation levels were decreased on the blended nanofiber surfaces, presumably due to their increased hydrophobicity. These results suggest that chitosan/oleamide nanofibers with high spinnability can be applied to the preparation of wound dressing membranes or patches with intrinsic antibacterial properties and moderate hydrophobicity. We expect that oleamide, which has lubricant and antibacterial properties, can be utilized as a blending component of chitosan-based nanofibers for biomaterial and tissue engineering applications.