Heterogeneous catalysts that activate peroxymonosulfate (PMS) are highly desirable in advanced oxidation process. In this study, Mo/Fe-S was synthesized from Mo/Fe-metal organic framework and used as PMS activator to inhibit bacterial growth and microbial attachment. The Mo/Fe-S material, prepared with a molar ratio of Mo:Fe = 1:1 (short for Mo/Fe-S), exhibited the highest number of S vacancies (SVs) and exposed the most active sites of Mo and Fe, which were responsible for the decomposition of PMS. Confirmed by systematic characterizations and theoretical calculations, SVs in Mo/Fe-S have strong adsorption energy for PMS. SO4− and OH are capable of damaging sulfate-reducing bacteria and Pseudomonas aeruginosa. At the metal sites, active species are produced through dual pathways involving the transfer of electrons in Fe(II)/Fe(III) and Mo(III)/Mo(IV). The interfacial reaction mechanism was investigated through in situ tests, and a mechanistic proposal based on vacancy catalysis and bimetallic active sites was introduced. This study provides new insights on polymetallic sulfides assisted with SVs in PMS activation for advanced oxidation processes.