In this study, in situ glyoxal crosslinked chitosan/poly (vinyl alcohol) (PVA) hydrogel nanofibers reinforced with halloysite nanotubes (HNT) were prepared by the electrospinning method without needing post-treatment for stabilization of the nanofibers in aqueous media. FTIR spectroscopy approved the formation of acetal bonds between glyoxal and hydroxyl groups of PVA and chitosan. Morphological studies by SEM/EDX and TEM in accordance with XRD patterns proved that HNT was successfully incorporated into the crosslinked chitosan/PVA nanofibers. The crosslinked nanofibers were insoluble in water. Due to the hydrophilic nature of HNT, the swelling of the nanofibers was increased from 272% for crosslinked chitosan/PVA nanofibers to around 400% for the HNT reinforced nanocomposite nanofibers. Comparing to chitosan/PVA nanofibers, the tensile strength of the crosslinked nanocomposite nanofibers was increased to 2.4 and 3.5 fold by incorporation of 3 and 5% HNT, respectively. Presence of HNT in chitosan/PVA nanofibers reduced the contact angle with water and increased the hydrophilicity of HNT-reinforced nanofibers favoring the attachment of fibroblast cells. Cytotoxicity studies by AlamarBlue assay showed that presence of HNT increased the biocompatibility of the nanofibers. It was also concluded that glyoxal can be used safely for crosslinking of chitosan/PVA nanofibers without any cytotoxic effect for fibroblast cells. From the results of this work, HNT reinforced chitosan/PVA nanofibers crosslinked by glyoxal are introduced as promising nanomaterials for skin tissue regeneration.