The food industry must prevent food contamination caused by pathogenic Escherichia coli strains as they pose a severe public health threat worldwide and lead to unimaginable economic losses. In this study, the synergistic antibacterial activity in vitro of nisin and phytic acid (PA), a natural metal chelate, against 5 foodborne isolates of E. coli were evaluated by a checkerboard assay. Checkerboard assay showed that nisin and PA showed a synergistic effect (FICI <0.375) on three O157 serogroup strains and an addictive effect (0.5<FICI <0.5625) on the remaining two non-O157 serogroup strains. Then the combined bactericidal effect of these compounds against E. coli O157:H7 was investigated. Results showed the number of planktonic and biofilm cells inactivated in groups treated with a combination of 0.512 mg/mL nisin plus 1 × MIC or 2 × MIC PA was significantly higher than the sum of groups treated alone (P < 0.05). Electron scanning microscopy studies revealed that the nisin plus PA treatment had a severe effect on E. coli O157:H7, which after treatment cell morphology appeared deflated with large invaginations. The effect of nisin plus PA against E. coli O157:H7 was furthermore assessed on cold-stored beef, where a similar synergistic bactericidal effect was observed. The studies demonstrate the great potential of the food-approved nisin to control the growth of undesired E. coli pathogens in foods. We used PA to overcome the permeability barrier of the outer membrane to nisin, but other food-grade metal ion chelators such as polyphosphate most likely could be used as well.