Phenyllactic acid (PLA), a promising food preservative, is safe and effective against a broad spectrum of food-borne pathogens. However, its mechanisms against toxigenic fungi are still poorly understood. In this study, we applied physicochemical, morphological, metabolomics, and transcriptomics analyses to investigate the activity and mechanism of PLA inhibition of a typical food-contaminating mold,Aspergillus flavus. The results showed that PLA effectively inhibited the growth ofA. flavusspores and reduced aflatoxin B1(AFB1) production by downregulating key genes associated with AFB1biosynthesis. Propidium iodide staining and transmission electron microscopy analysis demonstrated a dose-dependent disruption of the integrity and morphology of theA. flavusspore cell membrane by PLA. Multi-omics analyses showed that subinhibitory concentrations of PLA induced significant changes inA. flavusspores at the transcriptional and metabolic levels, as 980 genes and 30 metabolites were differentially expressed. Moreover, KEGG pathway enrichment analysis indicated PLA-induced cell membrane damage, energy-metabolism disruption, and central-dogma abnormality inA. flavusspores. The results provided new insights into the anti-A. flavusand -AFB1mechanisms of PLA.