Evaluation of the overall viability drop of bacteria within a box-type chamber is difficult because of a rapid decrease in the number of bacteria caused by collision with the wall/fan and natural decrease. In this study I aimed to develop and characterize a 1 m(3) airtight chamber and to investigate its suitability for evaluating the antibacterial activity of natural essential oils. Several circulation fans were placed in different locations within a 1-m(3) airtight chamber and were numerically simulated to determine the best conditions under which the residence time of circulated particles was the longest. The chamber was then fabricated based on the results of these numerical analyses. In experiments with Staphylococcus aureus, 6.6% (+/- 2.6%) of the bacteria were alive in the chamber when the bacteria were sampled for 20 min immediately after being injected for 20 min. To calculate the natural decrease in the number of injected bacteria, the test was repeated 10 times, and the average decrease was 2.1 x 10(6) CFU/mL In addition, obvious differences between the viability of bacteria in the presence and absence of antibacterial thyme oil were observed at 5- and 10-min intervals in the 20-min periods of injection and bacterial sampling. Various concentrations of thyme oil were used, and the time taken for the reaction between thyme oil and bacteria was calculated to determine spatial antibacterial activity. Each test resulted in 99.77% (+/- 0.02%) bacterial growth inhibition with 100% thyme oil, and 99.59% (+/- 0.58%) bacterial growth inhibition was observed after 10 min of reaction time. These test results show that the chamber was successfully designed for antibacterial evaluation testing. (C) 2012 Elsevier Ltd. All rights reserved.