Bacillus subtilis shows high contamination and antibiotic resistance in the food industry due to their biofilm‐forming capacity. This study aimed to search a biopreservative to inhibit the biofilm formation of B. subtilis. The effect of garlic extracts, nisin, ε‐polylysine, and citric acid on bacterial growth, cell surface hydrophobicity, and cell morphology were observed to provide insight into the antibiofilm mechanisms. Results showed that nisin, ε‐polylysine, and citric acid completely inhibited the growth and biofilm formation of B. subtilis at 0.15, 0.09, and 1.5 mg/ml, respectively. Garlic extracts inhibited cell elongation, but cannot completely inhibit the biofilm formation even at 1.8 mg/ml. The antibiofilm activities of four biopreservatives were due to antibacterial activities, reduction in extracellular polymeric substances production, and cell surface hydrophobicity. Among these four biopreservatives, ε‐polylysine, which caused cell to collapse, had the highest antibacterial and antibiofilm activities followed by nisin, citric acid, and garlic extracts. Practical applications Bacillus subtilis shows high contamination and antibiotic resistance due to their biofilm‐forming capacity. Finding a biopreservative to inhibit the growth and biofilm formation of B. subtilis was important in food industry. This study could pave the way for controlling B. subtilis in planktonic and biofilm states.