Coptis chinensis (CC) is used in conventional Chinese medicine. The main active components of CC are isoquinoline alkaloids, including berberine, coptisine, palmatine, and magnoflorine; all these are known to have several pharmacological properties. Poly(vinyl alcohol) (PVA) is a well-known synthetic biocompatible polymer suitable for a range of pharmaceutical uses; it can be used as a matrix for the incorporation of functional materials and has a wide range of applications in the cosmetics, food, pharmaceutical, and packaging industries. In this study, PVA-based electrospun nanofibers containing CC extract were successfully fabricated. Furthermore, the effects of different CC extract contents on the morphologies, and antimicrobial and antifungal properties of PVA/CC extract nanofibers were investigated. Morphological changes were observed using different molecular weights of PVA. For characterization, field-emission scanning electron microscopy, thermogravimetric analysis, and Fourier transform infrared analysis were performed. The effectiveness of these nanofibers has been demonstrated by evaluating the thermal stability against Staphylococcus aureus, antimicrobial activity against Staphylococcus aureus and Staphylococcus epidermidis, and the antifungal activity against the fungi Aureobasidium pullulans and Penicillium pinophilum. The PVA/CC extract nanofibers were found to have excellent antibacterial and antifungal activity and thermal stability; hence, their use in medicinal sectors is highly recommended.