A diverse community of microorganisms governs soil processes. Revealing changes in soil biota induced by management may help the development of management strategies to improve the productivity and sustainability of soil ecosystems. The main objective was to evaluate the effects of long-term management practices on the diversity and structure of the soil microbial communities as determined by Fatty Acid Methyl Ester (FAME) analysis. Five long-term (more than 30 years) treatments were evaluated, including undisturbed, set-aside from cultivation, moderately grazed, heavily grazed, and winter wheat (Triticum aestivum L.). The non-cultivated systems had the highest microbial biomass and the highest proportions of fungal and protozoan biomarkers. The undisturbed system had higher proportion of Gram-positive bacteria, while the grazed systems favored fast growing microorganisms such as Gram-negative bacteria. In the cultivated system, the microbial community also was dominated by Gram-negative bacteria, and higher proportions of cyclopropyl fatty acids that indicated nutritional stress. The correlations between enzyme activities and microbial biomass were stronger than between enzyme activities and phenotypic groups of organisms (Gram-positive and Gram-negative bacteria, actinomycetes, fungi, and protozoa), suggesting that the size of the microbial community rather than its composition had more impact on the enzyme functional capacity of the soil ecosystem.