The soil functioning and the response of the biota to external perturbations such as organic input are based on multitrophic interactions among a wide range of organisms. However, the various components of the soil microflora and microfauna are rarely addressed in ecological studies. We have developed a molecular approach based on terminal restriction fragment length polymorphism (T-RFLP) analysis to assess the community structures of protozoa and nematodes, together with bacteria and fungi that share the same soil environment. Two soils were characterized by a specific fingerprint for each of the four groups of organisms, showing the potential of all T-RFLP procedures to differentiate the community structures. The response of the soil biota to organic inputs was addressed using T-RFLP fingerprints together with physiological profiles of bacteria communities and global microbial activities and densities. Although the impact of compost or manure on the soil biota was only slightly noticeable from the global parameters measured, it was obvious from the community level analyses. However, the different components of the soil biota were altered to various extents, depending on the group of organisms and the soil–organic matter combination. The potential of the T-RFLP strategy to analyze simultaneously different biotic groups from the same soil DNA extract will facilitate the more systematic integration of eukaryotic organisms in ecological studies to investigate multitrophic interactions among the microflora and microfauna in relation with soil processes.