Staphylococcus aureus ATCC 25923 could form biofilm in the medium containing glucose (1%) and K2HPO4 (0.1 %). When Lactobacillus was incubated with planktonic S. aureus at the same time, Lactobacillus formed biofilm together with S. aureus and reduce biofilm formation about 15% on polystyrene surface of 96 well plates, consequently. However, if Lactobacillus was added into S. aureus biofilm, Lactobacillus could reduce approximately 83% S. aureus biofilm. Therefore, the study warned us to be careful when use Lactobacillus in inhibition of S. aureus biofilm in order to eliminate biofilm effectively.