One of the greatest challenges in soil ecology is to understand the mechanisms underlying the composition of complex soil communities. The members of these communities are not evenly distributed in soils; instead, soil organisms interact dynamically with biotic and abiotic factors which distribute heterogeneously in soil. Root chemical traits are key factors shaping belowground rhizosphere communities, because they mediate species-specific interactions, affect organisms at multiple trophic levels and, consequently, diverse ecological processes. This review explores the role of root chemical traits in shaping belowground biotic interactions. We first summarize which soil organisms occur and interact over a distinct, vertical gradient in the rhizosphere, i.e., from taproots to fine roots and exudates. Then we explore whether the vertical interaction gradient between roots and soil biota co-occurs with gradients of root chemical traits. We particularly discuss whether and why allocations of defensive chemicals in roots are consistent with the optimal defence allocation theory, which provides a mechanistic understanding of specific detrimental interactions. In order to obtain more detailed data, appropriate sampling scales and methods are necessary to correctly recognize belowground biodiversity and biotic interactions mediated by root chemical traits. Metabolomic approaches which enable us to analyse and identify thousands of root chemicals at once, will help to identify those chemical root traits which are the most important in belowground biotic interactions.