The rhizosphere is a key zone for pollutant removal in treatment wetlands; therefore, studies on microbial activity may provide helpful information for a better understanding of purification processes. We studied microbial activity in a vertical-downflow constructed wetland system treating waste activated sludge with high organic matter concentrations, under Mediterranean climate. The aims of the work were to study the influence of (i) the presence of plants, (ii) the plant species (Phragmites australis Cav., Typha latifolia L., Iris pseudacorus L.), and (iii) the plant growth stage (plant senescence and plant fast growing stage) on total respiration rate and phosphatase activity in the substrate (intented here as the solid support on which the plants grow). The presence of plants had a positive influence on microbial activity, since substrate respiration and both acid and alkaline phosphatase activity were always higher in planted than in unplanted mesocosms. Among the three tested species, Phragmites was the one that most stimulated both substrate respiration rate and phosphatase activity, followed by Typha and Iris. These differences of microbial activity between mesocosms were corresponding to differences of removal efficiency. Substrate respiration and phosphatase activity were of similar magnitude at the two growth stages, while the stimulating effect of plants seemed to have been delayed and microbial activity showed higher fluctuations at plant fast growing stage than at plant senescence.