Even though major adverse health conditions, such as obesity and Crohn’s disease, are tied to the human upper gastrointestinal (GI) tract, much less is known about this region compared to the lower GI tract. Thus, developing a deeper understanding of how chemical and microbial factors interact in the upper GI tract is essential. By using a multi-port catheter to sample stomach, duodenal, and multiple jejunal sites, we obtained GI fluid from healthy fasted and fed volunteers (N=26, 37 total admissions) at several timepoints over the course of 7 hours. We measured bile salt concentrations, 16S rRNA-gene-based microbial composition, and Clostridioides difficile str. R20291 germination efficiency in the GI fluid we collected. Primary and secondary conjugated bile salts predominated in the small intestine, with small amounts of unconjugated forms. These small concentrations of unconjugated bile salts positively correlated with the abundance of several bacterial taxa known to possess bile salt hydrolase. The high concentrations of conjugated bile salts promoted robust C. difficile germination across the upper GI tract. In this study, we further discuss relationships between bile salts and the indigenous microbiota in the human upper GI tract as well as the implications these findings have for C. difficile infection.