High Andean Páramos are very fragile neotropical ecosystems. Moreover, biodiversity in these areas is threatened by the anthropic activities of agriculture, cattle raising, and mining and has been little studied. Changes associated with potato farming and livestock grazing on microorganisms of Páramo soil of the Nevados National Natural Park (Nevados NNP) were assessed by (1) determination of physical and chemical properties (physicochemical matrix) and enzymatic activities associated (enzymatic activity matrix) with different biogeochemical cycles (C, N, and P); (2) microbial community functional diversity via evaluation of functional groups associated with carbon, nitrogen, and phosphorus cycles using cultivation-dependent techniques (arable functional group matrix and most probable number matrix); and (3) microbial diversity using cultivation-independent techniques that employ the hypervariable V5–V6 region of the 16S rRNA gene and pyrosequencing (16S-454 genus matrix and 16S-454 OTU matrix). Four of the six evaluated matrices (physicochemical, enzymatic activities, most probable number, and arable functional groups) revealed significant differences according to land use. The strategy adopted by the arable functional group matrix, in which the diversity of nitrogen fixation, phosphate solubilization, and cellulolytic compounds was evaluated, showed the most significant impacts of the different factors (land use, season, and elevation), especially those caused by potato cultivation and livestock. These results indicate that the initial impacts of potato farming and livestock grazing on the microbial community in El Bosque Village are better detected by functional diversity analysis than by molecular analysis of the 16S rRNA gene V5–V6 variable region. The results may have been caused by the type of molecular marker used in the analyses and the type of agricultural practices used by peasant farmers, which affect the functional diversity of the soil community. Among these practices are the maintenance of fallow periods greater than 7 years between each potato crop and the small proportion of cattle in relation to the total land area of the village. As the findings can be interpreted as an indicator of the early impacts of potato cultivation and livestock on microbial diversity, the effects of implementing community management plans, applying agroecological models, retaining biocultural memory, and changing agrarian structure are relevant for mitigating future changes.