We analysed two sites within and outside an urban development in a rural background to estimate the fungal richness, diversity and community composition in Quercus spp. ectomycorrhizas using massively parallel 454-sequencing in combination with DNA-tagging. Our analyses indicated that shallow sequencing (∼150 sequences) of a large number of samples (192 in total) provided data that allowed identification of seasonal trends within the fungal communities: putative root-associated antagonists and saprobes that were abundant early in the growing season were replaced by common ectomycorrhizal fungi in the course of the growing season. Ordination analyses identified a number of factors that were correlated with the observed communities including host species as well as soil organic matter, nutrient and heavy metal enrichment. Overall, our application of the high throughput 454 sequencing provided an expedient means for characterization of fungal communities.