Cinnamaldehyde and thymol are essential oils that are used as alternatives to antimicrobials in animal feed. The aim of the study was to examine the effects of cinnamaldehyde and thymol separately and in combination at amounts used in animal feed (5 mg · kg⁻¹ cinnamaldehyde and 15 mg · kg⁻¹ thymol) on cell membrane permeability, cell proliferation, tight junction integrity and cyclooxygenase-1 and -2 (COX1 and COX2, respectively) gene expression in the Caco-2 line, an intestinal epithelial cell model. Individually, thymol and cinnamaldehyde at below 50 mg · l−1 or 100 mg · l⁻¹ in cell culture medium exerted negligible effects on cell membrane permeabilization and proliferation. Thymol increased tight junction integrity by max. 61.7 ± 5.4% at 100 mg · ml⁻¹, whereas cinnamaldehyde weakened it by max. 76.8 ± 0.3% at 100 mg · l⁻¹. However, when the essential oils were combined together, tight junction integrity rose by 49.7 ± 4.4%, and no weakening effect of cinnamaldehyde was evident. Thymol up-regulated COX1 transcription and increased the COX1:COX2 ratio, which has been suggested to be beneficial for intestinal health. Treatment with combined essential oils for 48 h altered 33 genes expression by microarray analysis, with no enrichment in any gene ontology class. The combination of cinnamaldehyde and thymol did not affect membrane permeability or cell proliferation in intestinal epithelial cells. In contrast, it had beneficial effects on tight junction integrity. Thus, the combination of such essential oils as cinnamaldehyde and thymol at the amounts that are used in feed is not cytotoxic to Caco-2 cells, an intestinal epithelial cell model.