Electrospinning is a relatively simple technique to produce nanofiber mats, which can be used for diverse applications, from biomedicine to filtration to energy applications. For these cases, the large surface-to-volume ratio of nanofibrous membranes is often advantageous as compared to macroscopic textile fabrics or other structures. Additionally, the spinning process enables the integration of metallic or ceramic nanoparticles, blending different polymers or even preparing non-polymeric nanofibers by the calcination of the polymer used as a spinning agent. Especially for biomedical applications, the addition of an antibacterial agent can be supportive. Here, we report needleless electrospinning of nanofiber mats from poly(acrylonitrile) (PAN) with different mushroom mycelium powders, which have antibacterial and other positive properties. While PAN with Pleurotus ostreatus (oyster mushroom) powder could well be electrospun with the wire-based technique, PAN with Ganoderma lucidum (reishi mushroom) powder was nearly impossible to spin, with only one of four tests showing thin membranes containing fibrous as well as non-fibrous areas. The PAN/P. ostreatus nanofiber mats were further stabilized and carbonized. All samples were examined by confocal laser scanning microscopy (CLSM), scanning electron microscopy (SEM), and Raman microscopy, revealing similar morphology and carbon yield to pure PAN. This indicates the possibility to embed P. ostreatus powder in PAN nanofiber mats used for biotechnological or biomedical applications.