Stress significantly affects gastrointestinal and mental health, and the gut microbiota plays a pivotal role in this process. Enterococcus faecalis strain EC-12 (EC-12) is a lactic acid bacterium that has several health benefits. To investigate the impact of oral supplementation with heat-killed EC-12 on the discomfort caused by stress, a randomised, double-blind, placebo-controlled trial was conducted with students under academic stress taking EC-12 (n = 14) or a placebo (n = 13) daily for one week. Improvement in the students' symptoms was assessed using the visual analogue scale. Faecal microbiota was characterised by next-generation sequencing of 16S rRNA genes, and faecal metabolites and short-chain fatty acids were analysed using a GC-MS metabolomics approach. Significant improvements in abdominal pain and rumbling of the stomach were found in the EC-12 group compared to the placebo group, but no changes were observed in mental symptoms or salivary cortisol levels. The relative abundance of E. faecalis significantly increased in the EC-12 group after the trial; however, the composition and diversity of the gut microbiota did not change significantly. Functional analysis of the gut microbiota suggested that EC-12 intake alters specific metabolic pathways. Although the levels of faecal short-chain fatty acids did not change between the groups before and after the trial, EC-12 intake altered the composition of faecal metabolites, with a significant increase in tryptamine levels. The ratio of students with improved symptoms to those with increased tryptamine levels was calculated based on the number of students with elevated faecal tryptamine levels who showed symptomatic improvements. The ratio of improved rumbling stomach was higher than that of other types of digestive discomfort. These results suggest that oral supplementation with EC-12 has a potentially beneficial effect on stress-induced gastrointestinal discomfort, which may occur through alterations in gut microbiota composition and metabolism. This study was registered at the University Hospital Medical Information Network Center (UMIN) under the UMIN ID: UMIN000048184.