The antimicrobial efficacy of 400 nm photoirradiated caffeic acid (CA, 5 mM) was evaluated against Escherichia coli O157:H7 and Listeria innocua. A stronger antimicrobial effect was observed on E. coli than on L. innocua where the combined treatment resulted in 4 and 1 log CFU/mL reductions, respectively. The treatment’s effects on metabolism (resazurin assay), uptake of CA (fluorescence technique) and membrane damage (prodpidium iodide assay) were studied in both species. CA uptake increased in both species, but membrane damage was only observed in E. coli. The treatment had minimal impact on metabolic activity in both species. The treatment applied to the surface of spinach leaves was found to be effective against E. coli. A combined treatment of 400 nm light and plant extracts known to contain CA was also evaluated for antimicrobial activity against E. coli and found to be effective. The novel treatment proposed in this study has the potential to improve the microbial food safety of fresh produce.