A large proportion of humans live in urban environments where they spend the majority of their time indoors. Next-generation sequencing (NGS) methods have shown rapid colonization of diverse bacterial communities in built environments (BEs) are primarily shed from human bodies. However, the diversity of BE fungal assemblages, which has important impacts on human health, remain poorly understood. We applied NGS sequencing using the internal transcribed spacer (ITS) 1 region to investigate the diversity, source and temporal dynamics of fungi in restroom environments. Swab samples were collected weekly from surfaces in both male and female public restrooms over an 8-week period. Sequencing identified 2,550 unique species, and the majority of the identified fungi originated in the environment. Conversely, fungi commonly found on skin were scarce, despite the fact that bacteria assemblages from the same samples were dominated by bacteria commonly found on human skin. The fungal assemblages proved stable over time, indicating that this method can provide a baseline for monitoring fungal communities in BEs. NGS sequencing of the small subunit ribosomal RNA (rRNA) gene marker has become the gold standard for describing bacterial and archaeal diversity. Unfortunately, sequencing methods targeting the 18S rRNA gene have been less successful for fungal diversity because the 18S rRNA gene is not as variable in fungi. Amplification of the ITS region has improved fungal environmental research because the high variability among ITS sequences provides finer species resolution than the 18S rRNA gene. However, the high variability among ITS regions means that, unlike the rRNA gene sequences, it is not possible to align ITS sequences or make accurate phylogenetic trees across distantly related fungal phyla; ITS alignments among closely related fungi are still possible. Accurate phylogenetic trees are required for many microbial analysis tools. We thus present a bioinformatics tool called ghost-tree to create phylogenetic trees using fungal ITS regions. This tool combines 18S rRNA and ITS sequences to create a single phylogenetic tree that can be used for diversity analyses. The final tree is then constructed from ITS sequences of distantly related genera despite the fact that all ITS sequences cannot be aligned together.