Pullulan/clay (montmorillonite, MMT)/silver (Ag) hybrid nanofibers with variable Ag nanoparticle contents were fabricated by electrospinning from aqueous solution. The prepared nanofibers were characterized using field-emission scanning electron microscopy, transmission electron microscopy (TEM), and X-ray diffraction (XRD). In addition, the thermal stability of the nanofibers was investigated using thermogravimetric analysis. The XRD patterns and TEM images suggest the coexistence of pullulan, Ag, and MMT through intercalation of MMT layers in the hybrid nanofibers. Moreover, the shifting of the intercalated MMT XRD peak to the left with the addition of Ag nanoparticles indicates that Ag nanoparticles were introduced into the structure of the clays. The introduction of Ag nanoparticles increased the thermal stability and antibacterial efficacy of the pullulan/MMT/Ag hybrid nanofibers.