Palladium-modified nitrogen-doped titanium oxide (TiON/PdO) nanofibers were synthesized by a sol–gel process on activated carbon-coated glass fiber (ACGF) template. The TiON/PdO fiber was tested for visible-light-activated photocatalysis using Bacillus subtilis endospore as a target. Our work shows that TiON/PdO nanofibers display an effective antimicrobial effect on Bacillus spores. The palladium modification provides a strong enhancement over TiON in the photocatalytic disinfection. Microscopic characterization demonstrated that visible-light photocatalysis on TiON/PdO fiber caused drastic damage on the spore coat and internal structure.