Ergosterol is generally assumed to be one of the best indicators of fungal biomass and soil quality. The present case studies describe variations in ergosterol content in soil, sampled at three Danish farms. One is organically cultivated according to the biodynamical principles (ORG). The other two are conventionally cultivated, one with animal husbandry (CONV-H) and one with plant production (CONV-P). The analytical method combined extraction in a microwave oven, clean-up with a new SPE sorbent material (OasisTM), and quantification by HPLC. The average estimated concentrations of ergosterol were 1.66 μg g−1in ORG, 2.35 μg g−1in CONV-H and 1.13 μg g−1in CONV-P. These results correlated well with some of the important variables in the management systems of the soils (frequency of grass and clover, input of animal manure, total-C, frequency of ploughing). However, the exact causal relationships cannot be revealed using the case-study approach. The statistical analysis of the spatial variation in ergosterol across the field demonstrated very clearly that in some soils, small-scale (few cm2) variations in ergosterol concentrations may be quite large in comparison with large scale (few hundred m2) variations. This should be taken into account when planning the sampling strategy.