Fungal species have numerous important functions in the environment. Where these functions occur will depend on how fungi are spatially distributed, but spatial structures of fungal communities are largely unknown. This is especially true in hyperdiverse tropical tree canopy systems, which are understudied using high-throughput sequencing technology. Here we explore fungal communities in a Costa Rican tropical rainforest canopy, with a focus on local-scale spatial structure and substrate specificity of fungi. We sampled 135 locations across five tree branches and identified fungi from four substrate types: outer host tree bark, inner bark, dead bryophyte tissue, and living bryophytes. Samples were located between one centimeter and eight meters apart. Fungal community composition and diversity varied among substrate types, even when multiple substrates were in direct contact. Fungi were most diverse in living bryophytes, with 39% of all fungal OTUs found exclusively in this substrate, and the least diverse in inner bark. Fungal communities had significant positive spatial autocorrelation and distance decay of similarity only at distances less than one meter. Similarity among samples declines by half in less than ten centimeters, and even at these short distances, similarities are low with few OTUs shared among samples. These results indicate that community turnover is high and occurs at very small spatial scales, with any two locations sharing very few fungi in common. High heterogeneity of fungal communities in space and among substrates may have important implications for the distributions, population dynamics, and diversity of other tree canopy organisms, including epiphytic plants.