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Purpose: Prebiotics are defined as substances which selectively promote beneficial gut microbes leading to a health benefit for the host. Limited trials have been carried out investigating their effect on the microbiota composition of individuals afflicted by functional constipation with equivocal outcomes. In a 21-day randomised, controlled clinical trial involving 61 adults with functional constipation, a prebiotic formulation with partially hydrolysed guar gum and acacia gum as its main ingredients, significantly increased complete spontaneous bowel motions in the treatment group. This follow-up exploratory analysis investigated whether the prebiotic was associated with changes to the composition, richness, and diversity of the faecal microbiota. Methods: Participants provided a faecal specimen at baseline and on day 21 of the intervention period. Whole genome metagenomic shotgun sequencing comprehensively assessed taxonomic and functional composition of the microbiota. Results: Linear mixed effects regression models adjusted for potential confounders showed a significant reduction in species richness of 28.15 species (95% CI − 49.86, − 6.43) and Shannon diversity of 0.29 units (95% CI − 0.56, − 0.02) over the trial period in the prebiotic group. These changes were not observed in the control group, and functional composition was unchanged in both groups. Conclusion: In adults with functional constipation, the intake of a prebiotic formulation was associated with a decline of species richness and Shannon diversity. Further research regarding the associations between prebiotics and the composition and function of the gut microbiota is warranted. [ABSTRACT FROM AUTHOR]