The human gastrointestinal tract (GIT) is home to an abundance of diverse microorganisms, and the balance of this microbiome plays a vital role in maintaining a healthy GIT. The obstruction of the flow of bile into the duodenum, resulting in obstructive jaundice (OJ), has a major impact on the health of the affected individual. This study sought to identify changes in the duodenal microbiota in South African patients with OJ compared to those without this disorder. Mucosal biopsies were taken from the duodenum of nineteen jaundiced patients undergoing endoscopic retrograde cholangiopancreatography (ERCP) and nineteen control participants (non-jaundiced patients) undergoing gastroscopy. DNA extracted from the samples was subjected to 16S rRNA amplicon sequencing using the Ion S5 TM sequencing platform. Diversity metrics and statistical correlation analyses with the clinical data were performed to compare duodenal microbial communities in both groups. Differences in the mean distribution of the microbial communities in the jaundiced and non-jaundiced samples were observed; however, this difference did not reach statistical significance. Of note, there was a statistically significant difference between the mean distributions of bacteria comparing jaundiced patients with cholangitis to those without (p= 0.0026). On further subset analysis, a significant difference was observed between patients with benign (Cholelithiasis) and malignant disease, namely, head of pancreas (HOP) mass (p= 0.01). Beta diversity analyses further revealed a significant difference between patients with stone and non-stone related disease when factoring in the Campylobacter-Like Organisms (CLO) test status (p= 0.048). This study demonstrated a shift in the microbiota in jaundiced patients, especially considering some underlying conditions of the upper GI tract. Future studies should aim to verify these findings in a larger cohort.