Hydroxyl radical (•OH) and singlet oxygen (1O2) were detected by electron spin resonance (ESR) spectroscopy in a direct current He/O2 (2%) non‐thermal plasma microjet‐water system. ${}^{ \bullet }{\rm O}_{{\rm 2}}^{- } $ is shown to be the precursor of •OH. The concentrations of 1O2 and •OH are evaluated to be around 6 × 10−4 and 1.2 × 10−5 M, respectively. The survival rates of S. aureus exposed to plasma for 20 s in 1 ml H2O, SOD (100 U, for scavenging ${}^{ \bullet }{\rm O}_{{\rm 2}}^{- } $), D‐Man (0.15 M, for scavenging •OH), and L‐His (0.15 M, for scavenging •OH and 1O2) solutions were 0.7, 1.6, 13.4, and 40.9%, respectively, indicating that 1O2 contributes the most to the inactivation.