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Livestock production in Latin America has replaced tropical dry forests with conventional monocultures pastures (CP) that have degraded soils. As an alternative to CP, intensive silvopastoral systems (ISS) have been developed with multi-canopied vegetation that mimics native forest (F). The litter inputs and year-round presence of the tree rhizosphere in ISS, contribute to the formation of “fertile islands,” which is expected to impact biological activity and crop productivity. This study, investigated the impact of the conversion of CP to ISS, as well as the effect of canopy of Prosopis juliflora trees in a chronosequence of ISS (3–15years) on soil microbial communities and the physicochemical properties of soil. FAME (fatty acid methyl ester) profiles indicated that soil microbial community structure and composition shifted depending on land management systems. CP promoted the dominance of G⁻ bacteria, while ISS chronosequence and F favored actinomycetes and fungal biomass (total and arbuscular mycorrhizal fungi). In addition, soil microbial community (FAME profiles) of ISS chronosequence and F were more similar than with CP. An increase in the Cy/pre FAME ratio in CP suggested that the microbial community was under higher stress. The advantage of including trees in pasture systems was reinforced by the observation that FAME biomarkers, enzymatic activities and nutrient status were significantly higher beneath the canopy of P. juliflora. The results indicate that ISS are viable alternatives for improving soil quality and metabolic function, which is reflected in the significant increase in microbial biomass, FAME biomarkers and enzyme activities compared with CP.