Summary Background The gastrointestinal microbiota has an important role in mucosal immune homoeostasis and may contribute to maintaining mucosal healing in Crohn's disease (CD). Aim To identify changes in the microbiota, metabolome and protease activity associated with mucosal healing in established paediatric CD Methods Twenty‐five participants aged 3‐18 years with CD, disease duration of over 6 months, and maintenance treatment with biological therapy were recruited. They were divided into a low calprotectin group (faecal calprotectin <100 μg/g, “mucosal healing,” n = 11), and a high calprotectin group (faecal calprotectin >100 μg/g, “mucosal inflammation,” n = 11). 16S gene‐based metataxonomics, 1H‐NMR spectroscopy‐based metabolic profiling and protease activity assays were performed on stool samples. Results Relative abundance of Dialister species was six‐times greater in the low calprotectin group (q = 0.00999). Alpha and beta diversity, total protease activity and inferred metagenomic profiles did not differ between groups. Pentanoate (valerate) and lysine were principal discriminators in a machine‐learning model which differentiated high and low calprotectin samples using NMR spectra (R2 0.87, Q2 0.41). Mean relative concentration of pentanoate was 1.35‐times greater in the low calprotectin group (95% CI 1.03‐1.68, P = 0.036) and was positively correlated with Dialister. Mean relative concentration of lysine was 1.54‐times greater in the high calprotectin group (95% CI 1.05‐2.03, P = 0.028). Conclusions This multiomic study identified an increase in Dialister species and pentanoate, and a decrease in lysine, in patients with “mucosal healing.” It supports further investigation of these as potential novel therapeutic targets in CD.