The application of natural and innocuous compounds has potential in aquaculture as an alternative to antibiotics. We evaluated the effect of diet supplementation with Thymus vulgaris essential oil (TVEO) on the allochthonous microbial composition of rainbow trout. DNA was extracted directly from the intestinal contents, and the V3-V4 regions of the 16S rRNA genes were amplified by PCR. The bacterial composition was analysed using temporal temperature gradient electrophoresis (TTGE). No significant changes (P > 0.05) were detected in the TTGE profiles of TVEO-treated trout compared with the controls. The Dice similarity index revealed a high stability (C-s > 70%) of the intestinal microbiota in both groups during the 5-week period. Sequence analyses of the TTGE bands revealed the same bacterial composition in both groups, with most bacteria belonging to the Proteobacteria and Firmicutes phyla. The in vitro antibacterial activity of TVEO was assessed using a range of normal intestinal isolates and fish pathogens. The inhibitory concentrations for all the tested bacteria were higher than the TVEO levels used in trout, which may explain the in vivo results.