Escherichia coli O157:H7 is one of the major foodborne pathogenic bacterial that cause infectious diseases in humans. The previous found that a combination of kojic acid and tea polyphenols exhibited better activity against E. coli O157:H7 than using either alone. This study aimed to explore responses underlying the antibacterial mechanisms of kojic acid and tea polyphenols from the gene level. The functional enrichment analysis by comparing kojic acid and tea polyphenols individually or synergistically against E. coli O157:H7 found that acid resistance systems in kojic acid were activated, and the cell membrane and genomic DNA were destructed in the cells, resulting in “oxygen starvation”. The oxidative stress response triggered by tea polyphenols inhibited both sulfur uptake and the synthesis of ATP, which affected the bacteria's life metabolic process. Interestingly, we found that kojic acid combined with tea polyphenols hindered the uptake of iron that played an essential role in the synthesis of DNA, respiration, tricarboxylic acid cycle. The results suggested that the iron uptake pathways may represent a novel approach for kojic acid and tea polyphenols synergistically against E. coli O157:H7 and provided a theoretical basis for bacterial pathogen control in the food industry.