Microbial communities play a major role in terrestrial ecosystem functioning, but the determinates of their diversity and functional interactions are not well known. In this study, we explored leaf litter fungal diversity in a diverse Panama lowland tropical forest in which a replicated factorial N, P, K and micronutrient fertilization experiment of 40 × 40 m plots had been ongoing for nine years. We extracted DNA from leaf litter samples and used fungal-specific amplification and a 454 pyrosequencing approach to sequence two loci, the nuclear ribosomal internal transcribed spacer (ITS) region and the nuclear ribosomal large subunit (LSU) D1 region. Using a 95% sequence similarity threshold for ITS1 spacer recovered a total of 2523 OTUs, and the number of unique ITS1 OTUs per 0.5–1.0 g leaf litter sample ranged from 55 to 177. Ascomycota were the dominant phylum among the leaf litter fungi (71% of the OTUs), followed by Basidiomycota (26% of the OTUs). In contrast to our expectations based on temperate ecosystems, long-term addition of nutrients increased, rather than decreased, species richness relative to controls. Effect of individual nutrients was more subtle and seen primarily as changes in community compositions especially at lower taxonomic levels, rather than as significant changes in species richness. For example, plots receiving P tended to show a greater similarity in community composition compared to the other nutrient treatments, the +PK, +NK and +NPK plots appeared to be more dominated by the Nectriaceae than other treatments, and indicator species for particular nutrient combinations were identified.