Fungi and bacteria are the main decomposers of soil organic matter, however, in urban Technosols their role is still poorly understood. The respiration of active and potential active parts of fungi and bacteria was assessed for Technosols of Russian cities (Sergiev-Posad, Moscow, Kursk) compared to the undisturbed natural analogues (Retisols and Chernozems). The technique of determination of fungi and bacteria respiration applying selective antibiotics was adjusted for urban Technosols. The bactericide (streptomycin) applied for respiration inhibition in the Technosols should be added in the concentration by several thousand fold less (0.0005–0.3 mg g −1 ) than for the references (4–15 mg g −1 ). Such specific feature related to neutral and alkaline pH properties of urban soils (7.0–7.9). The fungi-to-bacteria ratio was found to be 1.4, 3.2 and 3.4 for Technosols of SergievPosad, Moscow and Kursk, respectively, and 3.4–3.9 for the references. In Technosols of Sergiev-Posad and Kursk the fungi and bacteria respiration after adding easily available nutrient substrate (glucose) decreased by 1.3–6.8 times compared to natural analogues. The differences in averaged fungal respiration between urban and natural soils were more explicit than for bacterial. Consequently, urbanization influences on the fungi and bacteria functioning related to soil organic matter decomposition: in urban soils it was significantly deteriorated. © 2020, Springer Nature Switzerland AG.