Analyses of phosholipid fatty acids (PLFA) and phospholipid etherlipids (PLEL) revealed differences in size and structure of microbial communities in the three soil zones of a potato field: ridge (RS), uncompacted interrow (IS), and tractor-compacted interrow soil (CS). The quantity of phosholipid biomarker concentrations (= microbial biomass) showed large differences among different zones, when lipid contents were related to fresh soil volume instead of soil dry matter. Compaction of interrow soil caused an increase in bacterial and eukaryotic biomass, expressed as total PLFA concentration, as well as an increase in total archaeal biomass, expressed as total PLEL concentration and caused a decrease in the fungi-to-bacteria ratio. Due to the higher waterfilled pore space (an indirect measure for reduced O2 availability) in CS, a more pronounced anaerobic microbial community was estimated than in IS, which serves as an explanation for the elevated N2O fluxes in this soil zone. Apart from the effect of O2 availability, microbial communities, especially populations of aerobic bacteria, ascinomycetes, fungi, algae, protozoa, and aerobic archaea responded to organic matter composition in the individual zones. Only in RS PLEL derived cyclic isoprenoids were found, which presumably indicate root-colonizing archaea. Following principal component analyses of specific biomarker profiles, the assumed substrate effect had the strongest influence on the differences in microbial community structure between the three soil zones.