Negatively charged phosvitin (PV) and positively charged chitosan (CS) were alternately deposited on negatively charged cellulose mats via layer-by-layer (LBL) self-assembly technique. The deposition of PV and CS was confirmed by X-ray photoelectron spectroscopy (XPS), Fourier transform infrared spectra (FT-IR), and wide-angle X-ray diffraction (XRD). Morphologies of the LBL films coating mats were observed by scanning electron microscope (SEM). Thermal degradation properties were investigated by differential scanning calorimetry (DSC) and thermo-gravimetric analysis (TGA). Additionally, the result of microbial inhibition assay indicated that the composite nanofibrous mats had excellent antibacterial activity against Escherichia coli and Staphylococcus aureus, which could be used for antimicrobial packing, tissue engineering, wound dressing, etc.