Microbiological and environmental variables involved in the removal of free sulfide were studied along an eutrophication transect in the Bassin d'Arcachon (France). At four sites, analyses were carried out on reduced sulfur compounds, iron species and total numbers of viable sulfur bacteria (sulfide-producing bacteria, colorless sulfur bacteria and purple sulfur bacteria). In addition, the chemical buffering capacity towards free sulfide and the potential microbiological sulfide oxidation rates were determined. In the ecosystem, no free sulfide occurs in the top layers of the sediment at all four sites, despite a high nutrient load and hence favourable conditions for sulfide-producing bacteria. The explanation of this apparent discrepancy was shown to be the high biological sulfide oxidizing capacity in combination with a high chemical buffering capacity. The data presented illustrate that the buffering capacity of sediments towards free sulfide is the combined result of the chemical and biological processes. The ratio between these were found to depend on the degree of eutrophication. It was shown that the chemical buffering capacity towards sulfide is severely overestimated when based on the pool of chemically reactive iron, a more realistic value is obtained by estimating the total amount of sulfide that can be added before free sulfide can be detected. A clear difference was observed between the numbers of colorless sulfur bacteria and the activity of the entire population. For a proper quantification of the sulfide buffering capacity of sediments, it is essential to estimate the concentration of iron and sulfur compounds that actually can react with sulfide, as well as to analyze the activities of sulfide-oxidizing microbes.