Bananas are a well-balanced typical prebiotic food rich in dietary fiber, oligosaccharides, vitamins, and minerals. However, evidence supporting the stress-relieving effects of banana consumption in daily life is scarce. Therefore, we investigated the effects of banana-intake on stress-related markers, such as the intestinal environment, biochemical markers, autonomic balance, and mood status, in an open, randomized, parallel-group controlled trial using 20 healthy adult volunteers divided into a banana-intake group (n = 13 subjects, two bananas daily for 2 weeks) and a non-intake control group (n = 7, no banana-intake for 2 weeks). We measured the intestinal environmental marker urinary indoxyl sulfate, stress markers (salivary cortisol and chromogranin A), autonomic nervous system activity markers (heart rate, natural logarithm of low-frequency power [LnLF], natural logarithm of high-frequency power [LnHF], and LnLF/LnHF ratio), and mood status before and after the 2-week experimental period. We assessed the rate of change before and after banana-intake for all parameters by performing comparisons between the banana-intake and the non-intake control group as well as between the effective and ineffective groups within the banana-intake group. There was no significant difference in the rate of change for all parameters before and after banana-intake compared with the non-intake group. However, approximately 50% of the banana-intake group showed decreased urinary indoxyl sulfate; decreased cortisol and chromogranin A levels; decreased heartbeat and LnLF power, increased LnHF power, and decreased LnLF/LnHF ratio; a decrease in the five negative subscales, an increase in the two positive subscales, and a decrease in total mood disorder score. Our findings suggest that banana-intake for 2 weeks improves the intestinal environment, leads to predominant parasympathetic activity, and provides stress relief and psychological stability in approximately 50% of healthy adults.