Abstract Extensive cattle production occupies more than 27% of the rural landscapes in Latin America, and nowadays is considered as a major driver of tropical deforestation. Conventional monoculture pasture (CP) systems have degraded soils and caused negative environmental impacts. Recent years have shown increasing interest in the development of alternatives to the CP with a high efficiency of internal resource use and thus lower input requirement and cost. To address this, intensive silvopastoral systems (ISS) have been developed where trees are integrated with crops, pastures, and cattle for environmental and economic benefits. Soil microbial communities drive nutrients cycling and play an essential role in maintaining and improving soil quality; however, little information is available on how ISS affects them. Therefore, the objective of this study was to investigate microbial (fatty acid community profiling and enzyme activities) and physicochemical (pH, total C, moisture, and bulk density) properties when CP are converted to ISS (n=3). The treatments (three replications) were: CP, native forest, and 12-15 years old ISS. The results shown that ISS reduced bulk density, penetration resistance, increased total C and soil microbial biomass (normalized on clay content), making them more effective C storage pools than CP. Principal component analysis revealed that enzymes activities were clustered according to land management practices, which suggest that these properties can be used as indicators of ecosystem disturbance. On the other hand, CP promoted dominance of Gram (-) bacteria, while adversely affects slow growing microorganisms such as fungi and actinomycetes that are known to be sensitive to disturbance. This study demonstrate that increases in microbial biomass and enzyme activities of C, N and P cycling in ISS that would represent positive changes in soil functioning compared with CP.