With the expansion of cities around the world there is a growing interest in the factors that influence biodiversity and ecosystem processes in urban areas. Fungi are exceptionally diverse and play key roles in ecosystem function, yet despite predictions of negative impacts due to urbanization, fungi have been generally overlooked in urban ecological studies. We surveyed fungi in 16 remnant river red gum (Eucalyptus camaldulensis: Myrtaceae) woodlands along a gradient of 4–35 km from the city of Melbourne (south-east Australia). Using both sporocarp surveys and terminal restriction fragment length polymorphism (T-RFLP; primer pair ITS1-F-ITS4), we examined relationships between fungal community composition, landscape context (i.e. urbanization) and soil physicochemical properties. Community compositions from sporocarp data were significantly correlated with those from T-RFLP data, largely because of correlations with ectomycorrhizal sporocarps (Spearman rank correlation coefficients ρ 0.31–0.42) rather than saprotrophic fungi (ρ 0.18–0.21). Principal components analysis of soil properties and non-metric multidimensional scaling ordinations of fungal community composition showed no clear separation of sites according to urbanization, and there were no significant correlations between fungal community composition and urbanization. However, fungal community composition was significantly correlated with soil chemical properties (ρ 0.41–0.55). These data suggest that site-scale soil properties, and associated effects of past and current land management activities, were more important in determining fungal community composition than the landscape-level influences of urbanization.