Peatland degradation is tightly connected to hydrological changes and microbial metabolism. To better understand these metabolism processes, more information is needed on how microbial communities and substrate cycling are affected by changing hydrological regimes. These activities should be imprinted in stable isotope bulk values (δ 15N, δ 13C) due to specific isotopic fractionation by different microbial communities, their metabolic pathways and nutrient sources. We hypothesize that stable isotope values and microbial abundance are correlated and act as indicators of different hydrological regimes. We sampled an East-West transect across European fens in 14 areas and conducted a stable isotope (δ 13C, δ 15N) and membrane fatty acid (mFA) analysis. Within each area an undrained, drained and rewetted site was selected. Rewetted sites were separated based on when rewetting occurred. We found differences in the upper layers of all sites in microbial-derived mFAs and stable isotope values corresponding to hydrological regimes. The highest and lowest quantities of microbial-derived mFAs were measured in undrained and drained sites, respectively. Fungal-derived mFAs were especially lower in drained sites. Simultaneously, δ15N stable isotope values were highest in drained sites. In addition, stable isotope values and microbial-derived mFAs showed distinct depth trends. In undrained sites stable isotopes values slightly increased with depth. In drained sites, δ15N values decreased downwards, whereas δ13C values increased. Overall microbial-derived mFAs decreased with depth. These patterns presumably result from anoxic conditions and high peat recalcitrance in the deeper layers. In sites with short time of rewetting, the microbial-derived mFAs and stable isotope values were similar to values of drained sites, while with increasing rewetting time values shifted to those of undrained sites. We conclude that biomarkers indicate that stable isotope values reflect specific microbial metabolic processes, which differ with hydrological regimes, and thus could indicate both drainage and rewetting in fens.