The assemblage of fungi including unicellular yeasts in schools is understudied. We conducted an environmental study to characterize fungal communities in classroom floor dust. We collected 500 samples from 50 elementary schools in Philadelphia, PA, and evaluated room dampness/mold conditions. Genomic DNA from dust was extracted for internal transcribed spacer 1 Illumina MiSeq sequencing to identify operational taxonomic units (OTUs) organized from DNA sequences. Differential abundance analyses were performed to examine significant differences in abundance among groups. We identified 724 genera from 1490 OTUs. The genus Epicoccum was not diverse but the most abundant (relative abundance = 18.9%). Fungi were less diverse but most dissimilar in composition in the most water-damaged classrooms compared to the least water-damaged, indicating differential effects of individual classroom water-damage on fungal compositions. We identified 62 yeast genera, representing 19.6% of DNA sequences. Cyberlindnera was the most abundant (6.1%), followed by Cryptococcus, Aureobasidium, Rhodotorula, and Candida. The average relative abundance of yeasts tended to increase with increasing dampness and mold score and was significantly (p-value = 0.048) higher in the most water-damaged classrooms (22.4%) than the least water-damaged classrooms (18.2%). Our study suggests the need for further research on the potential health effects associated with exposures to yeasts in schools.