A B S T R A C T Recent preoccupations regarding possible negative effects of pollution, inappropriate land management, climate change, desertification, erosion, compaction or over-exploitation on soils has led to initiatives for the survey of soils. One of them, EcoFINDERS, launched a pan-European survey in order to define normal operating ranges for soil biodiversity and quality, through the use of several methodologies. The objective of the current work was the characterization of European soils under different land uses and from representative bio-geographical locations, using a PLFA method developed in the context of the mentioned survey, in order to recognize and define operation ranges for soil PLFA. PLFA analysis demonstrated to be a valuable tool in the evaluation, comparison and distinction of several land uses and biogeographical (BG) zones. The analysis of the PLFA diversity with PCA and PERMANOVA/SIMPER demonstrated a good separation of soil samples relatively to each defined land use and BG zone. Soils of the same BG zone and land use group showed an average biomarker constitution with consistent differences of PLFA/biomarker compositions relatively to other groups, especially concerning land uses. Total PLFA biomass was consistently higher in non-arable environments, i.e., forest or grass soils. Arable soils contained a total PLFA biomass that was in average 2.5–2.9 times lower than grassland or forest soil. Arable and grassland soils contained significantly low relative quantities of the saprophytic Fungi biomarker 18:2v6,9c (which decreased more than 18:1v9c), and low Total Fungi biomarkers/Bacteria biomarkers ratio (TF/B). The opposite occurred in forestry soil samples, and the Boreal BG zone stood out, showing the highest total fungal composition, with a most important contribution of the 18:2v6,9c saprophytic Fungi biomarker. Grasslands (all BG zones) showed to be the richest (3.5–3.8%) in Arbuscular Mycorrhizae Fungi biomarker, 16:1v5c. High proportions of Gram-positive, relatively to Gram-negative biomarkers occurred essentially in arable soils, in this study, but also in Alpine grasslands, which main Gram-positive biomarkers were a15:0, and i15:0. Total biomass, PLFA Biomarker groups, and different balances between individual PLFA enabled the discrimination of samples originating from both different BG zones and land uses.