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Physical and chemical alterations may affect the microbiota of soils as much as the specific presence of toxic pollutants. The relationship between the microbial diversity patterns and the soil quality in a Mediterranean context is studied here to test the hypothesis that soil microbiota is strongly affected by the level of anthropogenic soil alteration. Our aim has been to determine the potential effect of organic matter loss and associated changes in soil microbiota of poorly evolved Mediterranean soils (Leptosols and Regosols) suffering anthropogenic stress (i.e., cropping and deforestation). The studied soils correspond to nine different sites which differed in some features, such as the parent material, vegetation cover, or soil use and types. A methodological approach has been used that combines the classical physical and chemical study of soils with molecular characterization of the microbial assemblages using specific primers for Bacteria, Archaea and ectomycorrhizal Fungi. In agreement with previous studies within the region, physical, chemical and biological characteristics of soils varied notably depending on these factors. Microbial biomass, soil organic matter, and moisture, decreased in soils as deforestation increased, even in those partially degraded to substitution shrubland. Major differences were observed in the microbial community structure between the mollic and rendzic Leptosols found in forest soils, and the skeletic and dolomitic Leptosols in substitute shrublands, as well as with the skeletic and dolomitic Leptosols and calcaric Regosols in dry croplands. Forest soils displayed a higher microbial richness (OTU’s number) and biomass, as well as more stable and connected ecological networks. Here, we point out how human activities such as agriculture and other effects of deforestation led to changes in soil properties, thus affecting its quality driving changes in their microbial diversity and biomass patterns. Our findings demonstrate the potential risk that the replacement of forest areas may have in the conservation of the soil’s microbiota pool, both active and passive, which are basic for the maintenance of biogeochemical processes.