Cambisols, Luvisols and Stagnosols are main soil taxonomical units covering 78% of the total area of the Czech Republic. In the period 2001-2008 soil samples from 13 arable and 2 grassed localities were collected and tested. Microbial biomass carbon (MBC) was determined using microwave irradiation method (MW), chloroform fumigation extraction method (FE) and re-hydration technique (RHD). Soil samples were moistened, according to the content of organic carbon (Corg), by deionized water addition (%Corg × 0.19 ml/g DM - dry mass of soil) immediately before MBC determination. Microwave sterilization (800 J/g DM = 600 W, 2 × 67 s, 100 g DM (10 soil samples) and microwave soil extracts digestion (800 J/ml = 250 W, 77 s, 24 ml) give the lowest values of MBC (204 ± 67 mg/kg DM; 100%) in comparison with FE (236 ± 57 mg/kg DM; 116%) and RHD (478 ± 138 mg/kg DM; 235%), respectively. High significant correlation (r = 0.9713) was found between TC (total carbon; 1.36 ± 0.29%) and TN (total nitrogen; 0.15 ± 0.03%) determined by CNS analyzer. Furthermore, high significant correlations were found between MBC-MW and MBC-RHD (r = 0.8965) as well as MBC-FE and DHA (dehydrogenase activity; r = 0.8094), respectively. DHA in studied soils reached 147 ± 68 mg of triphenylformazan/kg DM/24 h. Corg formed 96% of TC and total Kjeldahl nitrogen 97% of TN, respectively. According our results MW is fully acceptable for MBC determination.