Microbial inactivation by cold atmospheric plasmas has been a subject of tremendous research interest in recent years, in part, due to the ambiguity concerning the plasma factors responsible for bacterial inactivation. This work investigated the efficacy of an atmospheric‐pressure plasma jet ignited in either helium or helium/oxygen mixtures in inactivating Escherichia coli on agar. The correlation of data obtained from inactivation experiments and a 2D model describing the gas dynamics and afterglow chemistry showed that the inactivation mechanisms differed qualitatively between the two gas compositions. This work also provides insight into the reaction pathways that lead to the production and destruction of the key active species and illustrates the importance in these processes of admixing ambient air.