Many freshwater mussel species require fish hosts for successful reproduction; these relationships can be highly specialized and variable across mussel species. Most mussel host-identification studies have occurred in a laboratory setting and may miss important behavioral and spatial aspects of the mussel–host relationship. As such, conducting in situ evaluations of fish host use by mussel glochidia can provide additional information regarding fish–mussel relationships in the wild. However, in situ mussel–host examinations are rare, and few have been completed along the southern Atlantic slope of the United States. The goal of our study was to examine potential host use and glochidial-release timing in a small stream in South Carolina. Our objectives were to identify fish species used as mussel hosts, examine differences in the prevalence and intensity of infestations among host species, and quantify the effects of water temperature and stream flow on the probability of fish infestation. We examined wild-caught fishes for the presence of glochidial infestations in Sleepy Creek, a small tributary to Stevens Creek in the Savannah River basin of south-central South Carolina. We inspected a total of 822 individual fishes of 14 species and detected glochidia on 92 individuals of 5 fish species. Mean intensity of infestations varied from 7 to 45 glochidia per fish, but intensities did not differ significantly among fish species. Water temperatures and fish total length had significant positive effects on the probability of glochidial infestation. Highest glochidia prevalence occurred toward the end of our study at warmer temperatures and on mid-length fishes. Our study highlights the potential of in situ studies and DNA metabarcoding for identifying host use of wild glochidia.