Reactive oxygen species play important effects on organisms not only in vivo but also in vitro. The atomic oxygen radical anion (O-) has shown extremely high oxidation and reactivity towards small molecules of hydrocarbons. However, the O- effects on cells of microorganisms are scarcely investigated. This work showed the evidence that O- could react quickly with microorganisms (Escherichia coli, Bacillus subtilis, Staphylococcus aureus, Aspergillus niger, Saccharomyces cerevisiae, and Actinomycetes (5046)) and damaged the cell walls seriously as well as their intrinsic structures, arising a fast and irreversible inactivation. SEM and TEM micrographs were used to reveal the structure changes of cells before and after reacting with O- radicals. The inactivation efficiencies of the microorganisms depended on the O- intensity, the initial population of microorganisms, the exposed area, the environment, and the microorganisms’ types. Over 99% reduction of an initial 1.0×107 colony-forming unit (cfu), E. coli population only required less than 2 minutes while exposed to a 0.23 μA/cm2 O- flux under dry argon atmosphere (30°C, 1 atm). The observation of anionic intermediates (CO-, CO2-, H2O-, and anionic hydrocarbons) by time-of-flight (TOF) mass spectrometry and the neutral volatile products (CO, CO2, and H2O) by quadrupole mass spectrometry (Q-MS) provided an evidence of the reactions of O- with hydrocarbon bonds of the microorganisms. The inactivation mechanism of microorganisms induced by O- was discussed.