Information on microbial biomass carbon (MBC) is crucial to assess their stocks and role for plant nutrient release in soil. Next to fumigation-extraction, molecular methods are routinely used to estimate the contribution of fungi, bacteria, and archaea to the soil microbial community. However, more information on the links between these different indices would deepen the understanding of microbial processes. The current study is based on 11 datasets, which contain MBC and MBN data obtained by fumigation-extraction and information on bacterial, archaeal, and fungal gene abundance, totalling 765 data points from agricultural, forest, and rangeland soils. Some of these datasets additionally provide information on double-stranded deoxyribonucleic acid (dsDNA) and fungal ergosterol. MBC varied around the median of 206 µg g⁻¹ soil. MBN followed with a median MB-C/N ratio of 4.1. Median microbial gene abundance declined from bacteria (96 × 10⁸) to archaea (4.4 × 10⁸) to fungi (1.8 × 10⁸). The median ratio of MBC/dsDNA was 15.8 and that of bacteria/dsDNA was 5.8 × 10⁸ µg⁻¹. The relationships between MBC and dsDNA as well as between bacterial gene abundance and dsDNA were both negatively affected by soil pH and positively by clay content. The median ergosterol/MBC and fungi/ergosterol ratios were 0.20% and 4.7 (n × 10⁸ µg⁻¹), respectively. The relationship between fungal gene abundance and ergosterol was negatively affected by soil pH and clay content. Our study suggests that combining fumigation-extraction with molecular tools allows more precise insights on the physiological interactions of soil microorganisms with their surrounding environment.