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Phytoremediation was used to regenerate a limestone quarry area. Plant growth mixed medium added over the quarry surface, consisting of a mixture of pyrolusite byproducts, natural soil, sand, and rice husk. Three different plant species: pine, cypress, and broom were planted at 9 randomized plots in order to assess the effects of vegetation on the microbial development, which was measured for the following 3 years. Substrate samples were analyzed for organic carbon content (C), microbial biomass (C), basal CO respiration activity (BR), alkaline phosphatase (ALP), and acid phosphatase activities at each plant specie and year. Furthermore, the ratio C/C, the metabolic quotient (qCO), and the C mineralization quotient (qM) were determined. The highest survival rates occurred for broom (93.52%), followed by cypress and pine (82.41%) at the final year, while the content of C, BR, and ALP was increased significantly under plants (pine, cypress, and broom) compared with control. C content and BR was plant dependent. Cypress sites had the highest values of C (214.9 μgCg) and BR (112.8 μgCO-Cgd) at the 3 year. The plant root environment clearly enhances and regulates the microbial community, in correspondence to the species used. Below ground enhanced activity could fulfill the scope of phytoremediation strategies.