<p>Water-extractable organic matter (WEOM) obtained from air-dried soil samples can be used to estimate microbial biomass instead of chloroform fumigation extraction (CFE) using fresh, moist soils and toxic solvents. However, the accuracy of such WEOM-based estimates has not been evaluated. We evaluated relationships between WEOM measurements obtained from air-dried soils and microbial biomass measurements obtained through CFE based on 50 soil samples from 10 soil profiles in Japanese forests and pasture. The amount of water-extractable organic carbon (C) obtained from air-dried soils corresponded to 31% of microbial biomass C, demonstrating a strong correlation (squared correlation coefficients (<i>R</i>²) = 0.94, statistical probability (<i>P</i>) &lt; 0.01), whereas the amount of water-extractable total nitrogen (N) (WETN) and microbial biomass N was weakly correlated (<i>R</i>² = 0.56, <i>P</i> &lt; 0.01). Moreover, the relationships with soil physiochemical properties were similar between WEOC and microbial biomass C (<i>R</i>² = 1.00, root mean square error (RMSE) = 0.04), whereas those were less similar between WETN and microbial biomass N (<i>R</i>² = 0.73, RMSE = 0.28). The decoupling of WETN for air-dried soils and microbial biomass N was attributed to differences in the amounts of inorganic N compounds (NO₃^– and NH₄⁺) in K₂SO₄ extracts from chloroform-fumigated and unfumigated soils compared to those in water extracts from air-dried soils. These results support our hypothesis that WEOM originates from and is a valid estimate of microbial biomass, particularly for C, offering a solution to the practical difficulties involved in measuring microbial biomass using the CFE. Nevertheless, a caution should be noted that this novel method is still empirical estimation without less mechanistic or process driven explanation. Thus, there is a need to validate this new estimation in other sets of soil samples.</p>