An extraction method was tested for biomonitoring the biofilm samples containing heavy metals. The fractionation of metal complexes was performed via C-18-HPLC-ICP-MS and MALDI-MS, respectively. The extraction power of some reagents was determined for the heavy metal extraction from biofilm samples collected in Erdemli coast in the Mediterranean Sea. The ammonium acetate solution giving the highest extraction results was found as a suitable extraction reagent. The concentration and pH of the ammonium acetate solution were optimized and found as 1 M and 5, respectively. The chromatograms of metal complexes with the C-18-HPLC-ICP-MS system were taken to determine the effect of the pH of the metal complexes. After performing the extraction, metal bounded biomolecules were characterized by MALDI-MS for the fractions in the C18-HPLC system. It was seen that ammonium acetate extraction (1M, pH 5) might be used in biomonitoring studies due to relatively simple procedure, short analysis period, and low cost. The evaluation of the applicability of the method in biomonitoring studies might be supported by further studies with biofilms having similar characteristics.