A microbioassay was developed for evaluating the in vitro antifungal activity of 30 preselected essential oils. A template based on 10 serial dilutions with eight replicates per dilution arranged on two 96-well ELISA plates was used as a reproducible and standardized design to identify the in vitro effectiveness of these essential oils against Penicillium italicum Wehmer, Penicillium digitatum Sacc. and Colletotrichum musea (Berk. & M.A. Curtis) Arx, three postharvest fungal pathogens, on fruits. Growth of mycelium was monitored by measuring optical density (492 nm). Cinnamomum zeylanicum, Cinnamomum verum and Eugenia caryophyllus were found to be still active against all the three pathogens even at 100 ppm. Compared to other methods, this microbioassay proved to be a rapid, reproducible, and efficient method for testing the efficacy of essential oils that inhibit spore germination in P. italicum, P. digitatum and C. musea. The assay requires relatively small amounts of essential oils.