The effect of dietary FODMAP (fermentable oligo-, di- and mono-saccharides and polyols) in healthy adults is poorly documented. This study compared the specific effects of low and moderate FODMAP intake (relative to typical intake) on the faecal microbiome, participant-reported outcomes and gastrointestinal physiology. In a single-blind cross-over study, twenty-five healthy participants were randomised to one of two provided diets, ‘low’ (LFD) <4 g/d or ‘moderate’ (MFD) 14–18 g/d, for 3 weeks each, with ≥ 2-week washout between. Endpoints were assessed in the last week of each diet. The faecal bacterial/archaeal and fungal communities were characterised by eighteen participants from whom high-quality DNA was extracted by 16S rRNA and internal transcribed spacer 2 (ITS2) profiling and metagenomic sequencing. There were no differences in gastrointestinal or behavioural symptoms (fatigue, depression, anxiety) or faecal characteristics and biochemistry (including SCFA). Mean colonic transit time (telemetry) was 23 (95 % CI: 15, 30) h with the MFD compared with 34 (24, 44) h with LFD (n 12; P = 0·009). Fungal diversity (richness) increased in response to MFD, but the bacterial richness was reduced, coincident with the expansion of the relative abundances of Bifidobacterium, Anaerostipes and Eubacterium. Metagenomic analysis showed expansion of polyol-utilising Bifidobacteria and Anaerostipes with MFD. In conclusion, short-term alterations of FODMAP intake are not associated with symptomatic, stool or behavioural manifestations in healthy adults, but remarkable shifts within the bacterial and mycobiome populations were observed. These findings emphasise the need to quantitatively assess all microbial domains and their interrelationships to improve understanding of the consequences of diet on gut function.