Radio frequency (RF) heating has been used to study pasteurization technology, but its mechanism has not been thoroughly studied, especially for fungi. In this paper, pasteurization processes and potential bactericidal mechanisms of Aspergillus flavus spore using RF was investigated. The best heating and pasteurization efficiency were achieved at (Electrode gap 130 mm, conductivity of spore suspension 0.1 S/m, volume of spore suspension 40 mL). At the cellular level, with the increase of RF heating temperature to 80 °C, the number of surviving cells decreased dramatically, the degree of cellular damage deepened through observation, the absorbance values of nucleic acid and protein increased to 0.468 ± 0.016 and 0.526 ± 0.023 respectively, the conductivity reached 99.0 ± 0.5%, the fluorescence intensity of reactive oxygen content increased to 51.6 ± 1.6 AU, while adenosine triphosphate (ATP) concentration reduced to 0.57 ± 0.07 mM. At the molecular level, transcriptomics analysis showed that the number of differentially expressed genes (DEGs) increased with the temperature increased. Gene ontology (GO) annotation analysis showed that DEGs mainly existed in the molecular function. This study investigated the potential bactericidal mechanism of A. flavus using RF, provided some theoretical basis for the research of the pasteurization of fungi.