Atmospheric cold plasma (ACP) treatment is an emerging food technology for product safety and quality retention, shelf-life extension and sustainable processing. The activated chemical species of ACP can act rapidly against microorganisms without leaving chemical residues on food surfaces. The main objective of this study was to investigate the efficiency and mechanisms of inactivation of fungal spores and biofilms by ACP and to understand the effects of gas-mediated versus liquid-mediated mode of application against important fungal contaminants. Aspergillus flavus was selected as the model microorganism. A. flavus spores were exposed to either gas plasma (GP) or plasma activated water (PAW), whereas gas plasma alone was used to treat A. flavus biofilms. This study demonstrated that both GP and PAW treatment independently resulted in a significant decrease of A. flavus metabolic activity and spore counts with maximal reductions of 2.2 and 0.6 log10 units for GP and PAW, respectively. The characterization of the reactive oxygen and nitrogen species (RONS) in PAW and the spore suspensions indicated that the concentration of secondary reactive species was an important factor influencing the antimicrobial activity of the treatment. The biofilm study showed that GP had detrimental effects on biofilm structure, however, the initial inoculum concentration prior to biofilm formation can be a crucial factor influencing the fungicidal effects of ACP.IMPORTANCEThe production of mycotoxin-free food remains a challenge in both human and animal food chains. A. flavus, a mycotoxin-producing contaminant of economically important crops was selected as the model microorganism to investigate the efficiency and mechanisms of ACP technology against fungal contaminants of food. To our knowledge this is the first study directly comparing antifungal properties of gas plasma (GP) or plasma activated water (PAW) against fungi, as well as reporting the effects of ACP treatment on biofilms produced by A. flavus.