Different water treatment processes (physical and chemical) exist to obtain safe water for human or food industry supply. The advanced oxidation technologies are rising as a new alternative to eliminate undesirable chemicals and waterborne diseases. In this work, we analyze the power of the photo-assisted Fenton process using Fe(II)/H₂O₂ and UV radiation (365 nm) to inactivate Bacillus subtilis spores, considered among the most resistant biological structures known. Different concentrations of Fe(II), H₂O₂ and UV radiation (365 nm) were used to inactivate wt and some coat spore mutants of B. subtilis. Wt spores of B. subtilis were inactivated after 60 min using this process. In general, all defective coat mutants were more sensitive than the wt spores and, particularly, the double mutant was 10 folds more sensitive than others being inactivated during the first 10 minutes using soft reaction conditions. Presence of Fe(II) ions was found essential for spore inactivating process and, for those spores inactivated using the Fe(II)/H₂O₂ under UV radiation process, it is suggested that coat structures are important to their resistance to the treatment process. The photo-assisted Fenton process using Fe(II), H₂O₂ and UV radiation (365 nm) can be used to inactivate any water microorganisms with the same or less resistance that B. subtilis spores to produce safe drinking water in relatively short treatment time.