Soil enzymes mediate key processes and functions of the soils, such as organic matter decomposition and nutrient cycling in both natural and agricultural ecosystems. Here, we studied the activity of five extracellular soil enzymes involved in the C, N, and P-mineralizing process in both litter and surface soil layer of rainforest in the northwest region of the Colombian Amazon and the response of those soil enzymes to land use change. The experimental study design included six study sites for comparing long-term pasture systems to native forest and regeneration practices after pasture, within the main landscapes of the region, mountain and hill landscapes separately. Results showed considerable enzymatic activity in the litter layer of the forest, highlighting the vital role of this compartment in the nutrient cycling of low fertility soils from tropical regions. With the land use transition to pastures, changes in soil enzymatic activities were driven by the management of pastures, with SOC and N losses and reduced absolute activity of soil enzymes in long-term pastures under continuous grazing (25 years). However, the enzyme activities expressed per unit of SOC did not show changes in C and N-acquiring enzymes, suggesting a higher mineralization potential in pastures. Enzymatic stoichiometry analysis indicated a microbial P limitation that could lead to a high catabolic activity with a potential increase in the use of SOC by microbial communities in the search for P, thus affecting soil C sequestration, soil quality and the provision of soil-related ecosystem services.