Staphylococcus aureus is known for its resistance to antibiotic treatment as well as the ability to form biofilms. Biofilm formation has been seen in S. aureus infections, yet, the mechanism of biofilm formation is not completely understood. Many molecules, such as DNA and polysaccharides, have been identified in the biofilm microenvironment, but little is known about the enzymes involved in the process. In this paper, alkaline phosphatase (ALP) activity was investigated in S. aureus grown either in biofilm or suspension cultures, achieved using DNase I. A significant increase of ALP activity was observed in S. aureus biofilm culture compared to its suspension counterpart. Treatment of sodium orthovanadate, an ALP inhibitor, significantly decreased biofilm formation. Its inhibition was on par with DNase I treatment at specific doses. Thus, ALP may play an important role in the biofilm formation. Likewise, ALP inhibition may be a novel target for anti-biofilm therapeutics.