Tools that will enable the assessment of agricultural soil quality and include measurements of biological indicators, such as soil respiration or nitrogen mineralisation, are increasingly in demand. Such tools require the establishment of reference systems to provide comparative ‘baseline’ or ‘normal’ values. In this study, we measured the spatial and seasonal variability of eight biological indicators (including two eco-physiological quotients) in order to establish a reference system at the regional level of Wallonia (Southern Belgium). Respiration potential, microbial biomass carbon, microbial C/N ratio, net nitrogen mineralisation, metabolic potential of soil bacteria, earthworm abundance, microbial quotient, and metabolic quotient were measured at 60 sites across contrasting agricultural regions (different soil types and climate) in both grasslands and croplands. Additionally, the same biological indicators were measured four times during the vegetation period (April, June, August, and October) in 11 cropland sites to assess seasonal variability. Reference ranges were defined for each biological indicator, based on the addition of variances (seasonal and spatial) and the calculation of cumulative distribution functions. Land use was the most useful classification variable to define a reference system in Wallonia. Two separate reference systems, one for grasslands and one for croplands, were thus appropriate for Wallonia. Sampling season had a significant effect on all biological indicators. The inclusion of seasonal variability resulted in reference ranges 1.1–5.7 times wider than ranges accounting only for spatial variability. The reference system provides a basis for a first comparative assessment of soil quality for most agricultural soils of Wallonia, independent of sampling period.