Aims: To investigate the mode of action of a blend of essential oil compounds on the colonization of starch-rich substrates by rumen bacteria. Methods and Results: Starch-rich substrates were incubated, in nylon bags, in the rumen of sheep organized in a 4 x 4 latin square design and receiving a 60 : 40 silage : concentrate diet. The concentrate was either high or low in crude protein, and the diet was supplemented or not with a commercial blend of essential oil compounds (110 mg per day). The total genomic DNA was extracted from the residues in the bags. The total eubacterial DNA was quantified by real-time PCR and the proportion of Ruminobacter amylophilus, Streptococcus bovis and Prevotella bryantii was determined. Neither the supplementation with essential oil compounds nor the amount of crude protein affected the colonization of the substrates by the bacteria quantified. However, colonization was significantly affected by the substrate colonized. Conclusions: The effect of essential oils on the colonization of starch-rich substrates is not mediated through the selective inhibition of R. amylophilus. Significance and the Impact of the Study: This study enhances our understanding of the colonization of starch-rich substrates, as well as of the mode of action of the essential oils as rumen manipulating agents.