Abstract Methods for the measurement of the minimum inhibitory concentration (MIC) of antibacterial agents have been developed for water-soluble substances and require adaptation for use with water-insoluble essential oils. This paper reports a standardized agar dilution MIC method, using 0.5% v/v Tween 20 as a dispersant, which provides a reliable and reproducible technique. The method was tested using two Melaleuca alternifolia oil samples with two different levels of terpinen-4-ol (37% and 45% v/v). The MIC values of die tea tree oil samples against a wide selection of bacteria, moulds and yeast are reported.