Pesticides impact soil microorganisms in various ways. Despite the pivotal role of the latter in ecosystem functioning, the assessment of pesticides soil microbial toxicity is lagging behind the recent methodological advances in microbial ecology. We investigated the impact of nicosulfuron, a low dose sulfonylurea herbicide, on the structure, abundance and function of the soil microbial community using standardized methodologies (PLFAs, taxa-specific qPCR and enzyme activities). For this purpose a Tiered approach involving assessment i) at extreme, long term (five repeated application cycles) exposure schemes in a microcosm experiment conducted under greenhouse conditions (x0, x10, x100 and x1000 the recommended dose, Tier I) and ii) at realistic field exposure scenarios (x0, x1, x2 and x5, Tier II) was followed. Significant reductions in the abundance of Gram negative (beta-proteobacteria, planctomycetes) and Gram positive bacteria (actinobacteria) were indicated by both PLFA and qPCR analyses at low soil concentrations of nicosulfuron (0.25-1 mu g g(-1)), while a reduction of fungi at equally low levels of nicosulfuron in soil was found only by qPCR analysis. C- and P-cycling enzymes were particularly sensitive even at low soil concentration of the herbicide (0-1 mu g g(-1)). In contrast, no inhibitory effects of nic-osufluron at field conditions were found. The only exception was cellobiohydrolase which were impaired at herbicide rates higher than the recommended. We suggest that the use of a tiered microcosm-to-field experimentation combined with the application of standardized methodologies could provide a comprehensive assessment of the soil microbial toxicity of pesticides.