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The fungal loop model of semiarid ecosystems integrates microtopographic structures and pulse dynamics with key microbial processes. However limited data exist about the composition and structure of fungal communities in these ecosystems. The goal of this study was to characterize diversity and structure of soil fungal communities in a semiarid grassland. The effect of long-term nitrogen fertilization on fungi also was evaluated. Samples of rhizosphere (soil surrounding plant roots) and biological soil crust (BSC) were collected in central New Mexico, USA. DNA was amplified from the samples with fungal specific primers. Twelve clone libraries were generated with a total of 307 (78 operational taxonomic units, OTUs) and 324 sequences (67 OTUs) for BSC and rhizosphere respectively. Approximately 40% of soil OTUs were considered novel (less than 97% identity when compared to other sequences in NCBI using BLAST). The dominant organisms were dark-septate (melanized fungi) ascomycetes belonging to Pleosporales. Effects of N enrichment on fungi were not evident at the community level; however the abundance of unique sequences, sampling intensity and temporal variations may be uncovering the effect of N in composition and diversity of fungal communities. The fungal communities of rhizosphere soil and BSC overlapped substantially in composition, with a Jaccard abundance similarity index of 0.75. Further analyses are required to explore possible functions of the dominant species colonizing zones of semiarid grassland soils.