Bacteria can form biofilms, complex microbial communities protected from environmental stress, on food contact surfaces. Brassicaceae plant has been shown to contain bioactive compounds with antimicrobial activities. The objective of this study was to evaluate the synergistic effects of Brassicaceae species and proteinase K against E. coli O157:H7 biofilm. We determined the minimum biofilm inhibitory concentration, the fractional inhibitory concentration indexes, and the synergistic inhibitory effect of Raphanus sativus var. longipinnatus, R. sativus, and Brassica oleracea var. acephala extracts with proteinase K on E. coli O157:H7. The biofilm showed a 49% reduction with 2 mg/mL R. sativus. The combination of proteinase K 25 µg/mL significantly increased the effect of 2 mg/mL R. sativus var. longipinnatus and the combined treatment yielded up to 2.68 log reduction on stainless steel coupons. The results showed that the combination of R. sativus var. longipinnatus extract and proteinase K could serve as an anti-biofilm agent with synergistic effects for inhibiting E. coli O157:H7 biofilm on stainless steel surfaces.