Soil microbial properties are highly sensitive to present and past anthropogenic influences such as urban expansion, which is among the most drastic form of land-use change having substantial consequences for soils. New Moscow is an ambitious project to reduce the population of Moscow city that resulted in a rapid urbanization of former croplands, fallow lands, and forested areas. This study aimed to investigate the effects of historical land use in New Moscow on urban soil microbial properties. The land-use map of New Moscow from 2016 was compared with a similar map from 1981 to investigate the main urbanization pathways and land-use history of the new urbanized areas. Compared with 1981, the 2016 urban areas were more than three times greater, and the forest, pasture, and cropland areas were reduced by 9%, 87%, and 18%, respectively. Topsoil (0–10 cm) and subsoil (10–30 cm) samples were collected from 11 nonurbanized and 11 urbanized areas from current and former forest, pasture, and cropland soils. Soil microbial biomass carbon and respiration rates and chemical (pH and total C and N) and physical (bulk density and texture) properties were compared. Urban soils converted from forests and pastures showed negative effects of urbanization on topsoil microbial properties, whereas a conversion of cropland to urban soil increased microbial biomass carbon and microbial respiration. Soil microbial properties and N and C contents showed a strong correlation. The influence of historic land use on subsoil microbial properties was not significant.