The Biofilm of Staphylococcus aureus was tested against a various of substances at different concentrations for 12 hr., to show its stability. The chelating agent Ethylene diamine tetra acetic acid (EDTA) (1-5%) didn't show any effect on the biofilm stability. Oxidizing agents also didn`t show a negative effect on the biofilm stability. By adding enzymes like Pepsin and Papain (100-500µl) the stability of biofilm did not affect. Organic solvents e.g.; hexan, toluene and methyl alchohol added at 1-5% also did not destabilize the biofilm stability. Acids and basics like (HCl and NaOH) were added with concentrations of 1-5%. The effect of different substance on the stability of biofilm were very slight. Adjusting the bacterial media to pH 2-13 using HCl and NaOH before the biofilm formation was effected and showed positive effect and prevent biofilm formation between pH (2-5) and pH (10-13).