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Peroxynitrite is an important chemical in the human immune system, which has high biocidal activity and can resist the invasion of pathogens. Currently, in vitro generation of peroxynitrite faces major technological challenges, especially for gas‐phase production, which requires low‐pressure conditions. Here, we report the method of how to generate gas‐phase peroxynitrite at atmospheric pressure using dielectric barrier discharge. Results show that the peroxynitrite concentration positively correlates with energy density. Moreover, Penicillium digitatum, a common fungus contaminant greatly affecting food and fruit preservation, was used as a microbial pathogen model to confirm the biocidal effects of ozone‐free nitrogen oxides under different discharge conditions. The germination inhibition efficiency of P. digitatum is highly likely attributed to the synergistic effect of gaseous peroxynitrite and NO. [ABSTRACT FROM AUTHOR]