Soil is a key part of a biotope and microorganisms are dominant components contributing to soil functions. Conversely, established methods for valuation of biotopes according to Natura 2000 rely predominantly on the communities living on the surface. Here, we aimed to assess soil microbial biomass and community structure on five localities with range of biotope values by means of phospholipid fatty acid (PLFA) profiling. PLFA figures were affected both by sampling season (spring vs. autumn) and locality. In spring, the living microbial biomass (estimated by PLFA tot ) exhibited poor correlation to biotope values. These were, on the contrary, correlated to trans/cis PLFA, an indicator of microbial stress, (i.e., lower stress in higher-rated biotopes), and fungal/bacterial PLFA (i.e., higher-rated biotopes contained more fungi). The attempt to model biotope values from microbial characteristics explained a maximum of~50% of the variability; the best predictors were the trans/cis stress indicator, percentage of actinobacterial PLFA, and ratio of PLFA of Gram-positive to Gram-negative bacteria. These results show that soil microbial characteristics present partly new information and indicate the need to amend the procedures of biotope assessment. Soil PLFA profiling could serve as suitable methods for this purpose.