Introduction:This study was conducted to evaluate the effectiveness of the mycobiome diet (as presented in the book Total Gut Balance) on the human gut microbiome in general and the gut mycobiome (fungal community) in particular. Enrolled subjects were evaluated for improved health, gastrointestinal symptoms, and weight loss, as well as subjective reports of changes in energy, fatigue, and sleep. Method: Ten healthy volunteers (six males and four females ranging in age from 30 to 70) were enrolled in this 28day protocol. Participants completed a food journal, checking off daily and weekly required foods, as well as noting bowel movements, weight, and any digestive-related complications. Fecal samples were collected at the beginning and end of the study, with mycobiome and bacteriome profiles sequenced using ITS and 16S regions, respectively. Results: The mycobiome diet was highly successful at reducing pathogenic Candida species. Within two weeks, Candida species overall decreased by 72.4%; C. albicans in particular decreased 1.42-fold, while C. tropicalis was undetected after 4 weeks. Subjects significantly increased their levels of beneficial bacteria, specifically Faecalibacterium prausnitzii, Bifidobacterium, Roseburia, Lactobacillus, and Bacteroides. Furthermore, pathogenic bacteria decreased significantly, including Escherichia coli, Bacteroides fragilis, and Clostridium. The changes in the microbiome structure were accompanied with improvement in digestive symptoms, weight loss, less fatigue, more energy, better sleep, and fewer cravings for empty-calorie foods. Conclusion: Our data showed that adhering to the mycobiome diet for 4 weeks led to positive shifts in fungal and bacterial microbiome communities concurrently with positive improvement in GI symptoms and overall health.