Anthropogenic pressure is unavoidable for tropical ecosystems and the timely information of conservation decisions is a pressing issue in biodiverse, vulnerable, and understudied systems. Soil is a complex living system that both supports and retains the signature of the aboveground diversity. We assessed whether environmental DNA (eDNA) from soil can reveal changes in biodiversity along different ecological conditions in endangered tropical ecosystems to inform conservation policies. In mountain systems, we measured the diversity and turnover of bacteria, fungi, and eukarya communities along an elevational gradient to determine the transition between cloud forest and paramo ecosystems; being a subject of legal protection the latter one. In tropical dry forest, we measured the diversity and turnover of bacteria, fungi, and insect communities between perturbed and unperturbed localities to reveal how land use change impacts these “hidden” communities. In mountain systems we found a consistent decline of diversity with elevation for all the taxonomic groups, with variable rates of decline across biological groups. Most communities exhibited a marked turnover in composition between 3,400 and 3,500 m.a.s.l. In the case of perturbed/unperturbed dry forest, the alpha diversity of fungi and bacteria did not change with land use but there was a significant change in species composition. In particular, insect diversity decreased in perturbed areas and exhibited the highest turnover in community composition between perturbed/unperturbed areas. These results are a key step for understanding how land use change might impact diversity and overall ecosystem function. eDNA allows us to effectively survey changes in understudied components of biodiversity being a powerful tool for informing conservation planning needed for the persistence of sustainable tropical ecosystems.