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The objective of this study was to investigate whether preferential flow paths have higher microbial biomass and different microbial community structures than the rest of the soil. The organic C concentrations in the preferential flow paths were 10 to 70% higher than in the matrix. The organic N concentrations were also enriched in the preferential flow paths, as well as the effective cation exchange capacity and the base saturation. Microbial biomass was 9 to 92% higher in the preferential flow paths than in the matrix, probably due to the better nutrient and substrate supply. The DNA concentrations and direct cell counts showed a similar pattern, while domain-specific genetic fingerprints did not reflect the differences between flow regions. However, Pseudomonas displayed different population structures between preferential flow paths and soil matrix. This indicated that possibly only few populations with a broad acceptance for substrates and aerobic as well as anaerobic growth specifically profit from the favourable conditions in the preferential flow paths.