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Understanding bacterial adhesion to surfaces requires knowledge of the forces that govern bacterial–surface interactions. Biofilm formation on stainless steel 316 (SS316) by three bacterial species was investigated by examining surface force interaction between the cells and metal surface using atomic force microscopy (AFM). Bacterial–metal adhesion force was quantified at different surface delay time from 0 to 60s using AFM tip coated with three different bacterial species: Gram-negative Massilia timonae and Pseudomonas aeruginosa, and Gram-positive Bacillus subtilis. The results revealed that bacterial adhesion forces on SS316 surface by Gram-negative bacteria is higher (8.53±1.40nN and 7.88±0.94nN) when compared to Gram-positive bacteria (1.44±0.21nN). Physicochemical analysis on bacterial surface properties also revealed that M. timonae and P. aeruginosa showed higher hydrophobicity and …