Thymus daenensis is a native medicinal plant of Iran with high pharmacological effects. The present survey was performed to evaluate the antibacterial effects of T. daenensis extract toward Staphylococcus aureus, Listeria monocytogenes, Escherichia coli and Pseudomonas aeruginosa food-borne bacteria. Ethanolic extract of T. daenensis was prepared from plant's aerial parts. The diameters of the growth inhibition zones of bacteria against this extract and amoxicillin antibiotic disk was assessed by the disk diffusion test. Minimum inhibitory concentration was assessed using the broth micro-dilution method. The diameter of the growth inhibition zones of different concentrations of T. daenensis extract had the ranges between 7.00±0.54 to 15.00±1.09 mm. These findings revealed that the increase in the concentration of T. daenensis extract have caused significant increase in the diameter of the growth inhibition zone (P ≤0.05). Antibacterial effects of different concentrations of T. daenensis extract were lower than that of amoxicillin disk. T. daenensis extract had the highest antibacterial effects against P. aeruginosa. Additionally, T. daenensis extract had the highest antibacterial effects at lower concentrations (25 mg/mL) toward S. aureus. T. daenensis extract had the lowest MIC against S. aureus (6.00±1.00 mg/mL). The MIC of T. daenensis extract toward P. aeruginosa was 10.00±1.00 mg/mL. Antibacterial effects of T. daenensis ethanolic extract was determined in this survey. According to antibacterial effect of T. daenensis extract against food-borne bacteria, its application as a food preservative should potentially assess.