This study is focused on assessment of microbial community structure differences in urban soils by means of lipid analysis. Soil samples (0–20 cm deep topsoil layer) contaminated with trace metals to various extents were taken from six sites located in the rural part of the city of Kirov (Russia). The samples were measured for pH and total Pb, Ni, Cr, and Cd. To study the microbial community, microorganism markers (fatty acids) were extracted, which were then diagnosed by mass spectrometry. Nonmetric multidimensional scaling plots of soil communities showed relative dissimilarity in control and polluted soils. The total lipid content in control samples was found to be significantly higher than in polluted soil. The highest indicator value was assigned to Actinobacteria phylum, whose concentration decreased remarkably in polluted samples, and anaerobes Butyrivibrio sp. and Bifidobacterium sp. were regarded as an indicator for soils with relatively low pollution exposure. Microbial profiles were also indicative of selective enrichment with competent species (Desulfovibrio sp., Bacteroides fragilis, and Chlamydia sp.) in soil greatly contaminated with heavy metals. This study suggests that the method of lipid diagnostic can be highly indicative of soil microbial structure and thus it can be used as a quantitative measurement of urban soil biological quality.