Myrmecochory is a widespread mutualism in which plants benefit from seed dispersal services by ants. Ants might also be providing seeds with an additional byproduct benefit via reduced plant pathogen loads in the ant nest environment through their antimicrobial glandular secretions. We investigate this byproduct benefit by identifying fungal communities in ant nests and surrounding environments and quantifying fungal community change (1) through time, (2) between different nest substrates, and (3) as a function of average ant activity levels within nests (based on observed ant activity at nest entrances throughout the summer). We split fungal communities by functional guild to determine seed-dispersing ant-induced changes in the overall fungal community, the animal pathogen fungal community, the plant pathogen fungal community, and the myrmecochore pathogen fungal community. Nest substrate (soil or log) explained much of the variation in fungal community dissimilarity, while substrate occupation (ant nest or control sample) and time had no influence on fungal community composition. Average ant activity had no effect on the community turnover in fungal communities except for the myrmecochore pathogenic fungal community. In this community, higher ant activity throughout the summer resulted in more fluctuation in the pathogenic community in the ant nest. Our results are not consistent with a byproduct benefit framework in myrmecochory, but suggest that nest substrate drives dissimilarity in fungal communities. The influence of nest substrate on fungal communities has important implications for seeds taken into ant nests, as well as ant nest location choice by queens and during nest relocation.