The effects of food additives, including sucrose fatty acid ester with C18 (SFE C18) and monoglycerin fatty acid ester with C18 (MFE C18), gardenia yellow pigment (GY), monascus pigment (MP), milk serum protein (MSP), protamine (PT) and polylysine (PL) on biofilm formation by pathogenic and spoilage bacteria on stainless-steel were investigated. Salmonella Enteritidis, S. Typhimurium, Pseudomonas aeruginosa, P. fluorescens, Listeria monocytogenes and Staphylococcus aureus were cultivated with food additives at different concentrations, followed by evaluation of biofilm formation and viable counts after 24 or 48 h. SFE C18 inhibited biofilm formation by S. Enteritidis, S. Typhimurium and L. monocytogenes at 0.005% (w/w). The inhibition of biofilm formation by MFE C18 was weaker than that of SFE C18. GY reduced more than 50% of biofilm formation by S. Typhimurium even at 0.01%. MP greatly decreased biofilm formation of S. Enteritidis at 0.1%. MSP inhibited biofilm formation of S. Enteritidis and S. aureus at 0.0025%, but not those of S. Typhimurium, P. aeruginosa and L. monocytogenes at 0.025%. PT greatly reduced the biofilm formation of S. Enteritidis, S. Typhimurium and S. aureus with increasing PT concentration from 0.001 to 0.1%. PL inhibited biofilm formation on stainless steel by S. Enteritidis, S. Typhimurium, and L. monocytogenes with increasing PL concentration from 0.001 to 0.1%. In contrast, Biofilm formation of P. aeruginosa was promoted by GY, PT, and PL, that of S. aureus by MP, and P. fluorescens by PT at the concentrations effectively decreased those of the other bacteria. Especially, 0.1% PT and 0.01% PL killed planktonic cells of P. aeruginosa though increased the biofilm formation of the bacterium.