There is a growing interest for using natural preservatives in the food and dairy industries including the application of bacterial cultures to inhibit fungal spoilage. Several antifungal metabolites from bacteria have been identified, but their relative importance has been difficult to establish. In dynamic systems such as fermented milk products, the complexity of the food matrix affects detection, identification and quantification of antifungal metabolites, and thereby the understanding of the bacterial-fungal interactions. To ease the identification and quantification of bacterial metabolites (as judged by ultra-performance liquid chromatography/mass spectrometry) a chemically defined interaction medium (CDIM) was developed. The medium supported growth of antifungal cultures such as Lactobacillus paracasei and Propionibacterium freudenreichii, as well as spoilage moulds and yeasts isolated from fermented milk products. Both strong and weak antifungal interactions observed in milk could be reproduced in CDIM. The medium seems suitable for studying antifungal activity of bacterial cultures.