Background Studying the drivers and determinants of species, population and community spatial patterns is central to ecology. The observed structure of community assemblages is the result of deterministic abiotic (environmental constraints) and biotic factors (positive and negative species interactions), as well as stochastic colonization events (historical contingency). We analyzed the role of multi-scale spatial component of soil environmental variability in structuring earthworm assemblages in a gallery forest from the Colombian “Llanos”. We aimed to disentangle the spatial scales at which species assemblages are structured and determine whether these scales matched those expressed by soil environmental variables. We also tested the hypothesis of the “single tree effect” by exploring the spatial relationships between root-related variables and soil nutrient and physical variables in structuring earthworm assemblages. Multivariate ordination techniques and spatially explicit tools were used, namely cross-correlograms, Principal Coordinates of Neighbor Matrices (PCNM) and variation partitioning analyses. Results The relationship between the spatial organization of earthworm assemblages and soil environmental parameters revealed explicitly multi-scale responses. The soil environmental variables that explained nested population structures across the multi-spatial scale gradient differed for earthworms and assemblages at the very-fine- (<10 m) to medium-scale (10–20 m). The root traits were correlated with areas of high soil nutrient contents at a depth of 0–5 cm. Information on the scales of PCNM variables was obtained using variogram modeling. Based on the size of the plot, the PCNM variables were arbitrarily allocated to medium (>30 m), fine (10–20 m) and very fine scales (<10 m). Variation partitioning analysis revealed that the soil environmental variability explained from less than 1% to as much as 48% of the observed earthworm spatial variation. Conclusions A large proportion of the spatial variation did not depend on the soil environmental variability for certain species. This finding could indicate the influence of contagious biotic interactions, stochastic factors, or unmeasured relevant soil environmental variables.