In forests, the majority of fine roots are located within the upper soil horizons, and fine root biomass decreases with depth. We evaluated spatial patterns in the distribution of fine root biomass and determined relationships with soil properties and vegetation structure in a Eucalyptus tereticornis woodland in East Australia. Fine root biomass (0–50 cm depth) was 678 (± 96.9) g m−2 and decreased exponentially with depth. Total fine root biomass was positively related to aboveground herbaceous biomass and increased with increasing proximity to larger trees, reflecting contributions from both herbaceous understorey plants and mature trees. Plants produced more fine roots in soil patches with lower organic matter content, possibly as a functional response to increase acquisition of essential nutrients in more nutrient-depleted soils. Aboveground plant attributes were more important predictors of fine roots in the shallowest layer, while water availability was a stronger predictor of fine root biomass in deeper layers, likely reflecting the harsh climatic conditions prior to sampling. Fine roots represent an important gap in many ecosystem models despite being key for biogeochemical cycling. Here, we showed that the spatial patterns of fine root biomass can be inferred from soil and vegetation characteristics across remnant Australian Eucalyptus woodlands.