The objective of the present study was to evaluate the effect of a paediatric tube feed supplemented with a multifibre mixture on the gut microbiota and nutritional and micronutrient status of children on long-term enteral nutrition (EN). A randomised, controlled, double-blind, cross-over trial (2 × 3 months) with a washout period of 1 month was carried out. Twenty-seven children (80% neurologically impaired) aged 11.9 (SD 3.9) years, on long-term EN (4.8 (SD 3.9) years) were recruited. The analyses of the children's faecal pH, microbiota along with anthropometric measures, bowel movements and markers of blood micronutrient status were made. Twenty children completed the study. A significant increase in the proportion of stool bifidobacteria (+16.6%, P < 0.05) was observed during the multifibre period than during the fibre-free period, together with a significant reduction in stool pH (P < 0.001). Stool frequency and consistency as well as growth did not differ between the two periods. There was a significant increase (P < 0.05) in plasma ferritin at the end of the fibre-free period, but plasma ferritin levels remained within normal ranges during both periods. No diet effects on other blood parameters were observed. In conclusion, addition of a multifibre mixture with prebiotic components to paediatric EN is well tolerated, promotes bifidobacteria and reduces stool pH, indicating an improved gut health.