Wood is an important construction material, but a significant problem hindering its widespread use is susceptibility to biodeterioration and biodegradation. To protect wood against degradation, a surface coating can be used, and it is important to be able to predict the ability of the coating to prevent fungal growth. The currently available standard method to determine the antifungal efficiency of a coating has two weaknesses, viz. no evaluation of the moisture content in the wood material, and no possibility to study antifungal effect of the coating towards an individual fungus. A new quantitative method of determining the antifungal efficiency of coatings is therefore proposed, where a coating is applied to wood and exposed to an individual fungus in a Petri dish. Six commercial water-based coatings containing synthetic biocides were studied on filter paper (EN 15457) and with the new test method on wood blocks. The results show the importance of studying the antifungal efficiency of a coating using individual fungi instead of a mixture of fungi, since individual fungi interact differently with a given biocide in the coating. The moisture content of the wood substrate during the test was affected by how the fungus was established on the coating. This new test approach shows promise in screening the antifungal efficiency of wood coatings containing preservative substances applied to wood material surfaces.