The formation of biofilm by foodborne pathogens increases the risk of foodborne diseases, resulting in major health risks. Research on strategies for eliminating biofilm formation by foodborne pathogens is urgently needed. Therefore, the objective of this study was to construct a new technique for controlling foodborne bacteria and inhibiting the biosynthesis of biofilm via using natural products. The essential orange oil (EOO) and cell-free filtrate of Lactobacillus pentosus RS2 were used as antibacterial and antibiofilm agents against B. cereus RS1, the strongest biofilm-forming strain. The mixture of cell-free filtrate (CFF) and EOO (CFF/EOO) was the best antibiofilm agent under all tested conditions. The minimal inhibitory concentration (MIC) test revealed that 400 μl ml−1 CFF and 16 μl ml−1 EOO completely inhibited the growth of B. cereus. The treatment of three commercial surfaces with CFF/EOO resulted in a high reduction in biofilm synthesis, with adhesion percentages of 33.3, 36.3, and 40.8% on stainless steel, aluminum foil, and aluminum, respectively. The aluminum surface had the greatest adhesion with B. cereus RS1 among the three tested surfaces. These results were confirmed by expression analysis of three essential coding genes, sinR, calY, and spo0A, participating in biofilm formation in B. cereus. The biofilm-negative regulator gene sinR was overexpressed, whereas the biofilm-positive regulator genes calY and spo0A were down-expressed in B. cereus RS1 after treatment with antibiofilm agents, compared with those in the untreated sample. This study revealed that CFF/ EOO was more effective at activating sinR (2.099 ± 0.167-fold increase) and suppressing calY and spo0A (0.314 ± 0.058 and0.238 ± 0.04-fold decrease, respectively) compared to control. This result confirmed the biochemical estimation of biofilm formation in B. cereus after treatment with all the experimental agents. The EOO and CFF of L. pentosus RS2 can be used as strong antibacterial and antibiofilm agents against foodborne bacteria. These products reduced the biofilm formation on trade surfaces affecting the expression of three essential biofilm regulatory genes. This study considered novel research concerning the potential antibiofilm activity of EOO combined with CFF of L. pentosus and the molecular analysis of genes regulating biofilm production under stress of CFF/EOO.