In recent years, inactivation of pathogenic microorganisms by sulfate radical anion (SO4-center dot) has attracted increasing attention due to growing demands to control harmful disinfection byproducts and update water treatment systems for efficient microbial control. This critical review focuses on the basic principles and current research status of SO4-center dot-based inactivation technology, and for comparison includes (OH)-O-center dot-based inactivation of microorganisms. After a brief review of basic mechanisms of radical reactions with biomolecules, the inactivation kinetics and mechanisms by SO4-center dot in various activation systems are summarized and discussed. We demonstrated that SO4-center dot oxidatively damages the cell membrane/wall, proteins, and genetic materials (i.e., DNA and RNA), resulting in the inactivation of the microorganisms. Finally, existing problems, challenges, and possible solutions in engineering applications, and future research directions are discussed.