Naturally occurring many biological structures have provided sources of inspiration for the fabrication of many novel nanostructures for various applications. Electrospun nano/microfibrous structures have great potential as scaffolds for cell attachment and proliferation in the field of tissue engineering. Here, for the first time, we report on the preparation of three-dimensional (3D) fungal mycelial mats with chitin-glucan polysaccharide cell walls as nano/microfibrous scaffolds for tissue engineering applications. Treatment of fungal-scaffolds (F-scaffolds) with β-mercaptoethanol (BME) improved hemocompatibility, and conferred biocompatibility with respect to the adhesion and proliferation of human keratinocytes. Field-emission scanning electron microscopy (FE-SEM) of BME-treated F-scaffolds revealed a meshwork of nano- and micro-fibrous mycelial structures with an average diameter of 2.94 ± 0.96 μm (range 0.92-5.6 μm). Tensile testing showed F-scaffolds had a mean tensile strength of 0.192 ± 0.07 MPa and a mean elongation at break of 10.74 ± 2.53%, respectively. The degradation rate of the F-scaffolds showed ~19.2 ± 1.9% weight loss in 28 days. FE-SEM of BME-treated F-scaffolds seeded with keratinocytes showed deposition of extracellular matrix (ECM) components and the formation of cell sheets in 14 days. In addition, the in vitro cytocompatibility of BME-treated F-scaffolds with keratinocytes was analyzed using resazurin-based assay, which showed a time-dependent increase in metabolic activity up to culture day 21. Overall, this novel investigation shows that filamentous fungal mats with a nano/microfibrous mycelial architecture are potentially useful for tissue engineering applications.