Purpose: The production of Technosols is a sustainable strategy to reuse urban wastes and to regenerate degraded sites. However, little is known regarding the role of the activity of enzymes associated with carbon and nutrients cycling on organic degradation and microbial activity in these soils. Methods: A controlled experiment was conducted with Technosols made from construction wastes, wood chips, and compost or compost plus biochar, in order to evaluate their organic matter (OM) degradation potential and functioning through the activity of enzymes and microbial community composition. Results: The Technosols had organic carbon contents from 13 to 30 g kg−1, carbon-to-nitrogen ratio from 10 to 20, and available phosphorus from 92 to 376 mg kg−1. The Technosols with biochar and compost had alkaline pH and higher contents of organic carbon and available phosphorus compared to Technosols with compost alone. The mixture of wood chips and compost presented the highest enzyme activities, and might be the most appropriate for Technosol’s production. The mixture of concrete and excavation waste with compost and compost plus biochar displayed a potential for OM decomposition comparable to that of wood chips with compost plus biochar. These results suggest that the bacterial and archaeal fingerprint is similar among the Technosols, although differences are observed in the relative abundances of their taxa. Conclusions: Substrate composition affects the processes of OM transformation, microbial biomass activity, and composition. The mixture of wood chips and compost presented the highest enzyme activities during the incubation period, and might be the most appropriate for its application as a Technosol. The mixture of concrete and excavation waste with either compost or compost plus biochar displayed a potential for organic matter decomposition that was comparable to that of the mixture of wood chips with compost plus biochar. The microbial communities in these Technosols are not significantly different yet, but the bioavailability of nutrients derived from the changes in the soil matrix (by adding construction waste and biochar) is influencing soil enzymatic activity.