The aim of this work was to examine the effect of pine forests on the soil microbial community along an altitudinal gradient in the Cuenca Mountains, Spain. Six experimental forest areas and two tree diversity levels (monospecific and mixed pine forest) were selected according to the following types: (i) at lower altitudes (up to 960 m above sea level), a monospecific Spanish black pine (Pinus nigra Arn. ssp salzmannii) forest stand and a mixed forest stand (maritime pine (Pinus pinaster Ait.) and Spanish black pine); (ii) at medium altitudes (up to 1350 m), a monospecific Spanish black pine forest stand and a mixed forest stand (Scots pine (Pinus sylvestris L.) and Spanish black pine); and (iii) above 1670 m, a monospecific Spanish black pine forest stand and a mixed forest stand (Scots pine and Spanish black pine). Soil moisture and temperature and different forest stand variables such as tree species composition, tree height, basal area and shrub cover were measured. Physicochemical soil properties including texture, pH, carbonates, total organic carbon, organic matter, electrical conductivity, N, P, soil enzyme activities, soil respiration and soil microbial biomass carbon were analysed at the selected forest areas and tree diversity levels. Results showed that soil moisture and temperature differed significantly across the altitudinal gradient. Carbon:nitrogen ratio, total carbonates, total organic carbon, phosphorus and total N values also differed at each experimental area but tree diversity level was not an influential factor. Soil respiration, microbial biomass carbon and enzyme activities tended to be less at low altitudes whereas no differences were found between monospecific and mixed pine forests across the altitudinal gradient. This work suggests that the soils of the Cuenca Mountains may be more sensitive to some physical and chemical site-specific aspects such as soil temperature and moisture than changes in tree stand composition (when considering only pine species). In addition, differences in soil physicochemical properties found at each experimental area may also act as additional factors for regulating enzymatic activities.