A method has been developed for mercury(II) and methyl mercury speciation on Staphylococcus aureus loaded Dowex Optipore V-493 micro-column in the presented work, by using cold vapour atomic absorption spectrometry. Selective and sequential elution with 0.1molL −1 HCl for methyl mercury and 2molL −1 HCl for mercury(II) were performed at the pH range of 2–6. Optimal analytical conditions including pH, amounts of biosorbent, sample volumes were investigated. The detection limits of the analytes were 2.5ngL −1 for Hg(II) and 1.7ngL −1 for methyl mercury. The capacity of biosorbent for mercury(II) and methyl mercury was 6.5 and 5.4mgg −1 , respectively. The validation of the presented procedure is performed by the analysis of standard reference material. The speciation procedure established was successfully applied to the speciation of mercury(II) and methyl mercury in natural water and microwave digested fish samples.