Mold growth constitutes a problem in many food and clinical environments and there is therefore focus on studying antifungal activity. Methods for determining growth inhibition by measuring colony growth or biomass are, however, time-taking and rapid methods for evaluation of antifungal effects are needed. Propionic acid and diacetyl are antifungal compounds produced by a range of dairy-associated bacteria. Their activity against Penicillium spp. was monitored real-time using an optical detection system with tilted focus plane to assess growth and morphological changes of Penicillium spp. by image recording inside a 96 well microplate. Images were used for generation of growth curves by using a segmentation and extraction of surface areas (SESA) algorithm and for quantifying morphology changes. Using image analysis growth could be detected within 15 h compared with more than 30 h when using standard optical density measurements. Induced morphological changes of fungi could furthermore be visualized and quantified using morphological descriptors such as circularity, branch points, perimeter and area of spores and growing hyphae. Propionic acid inhibited two out of two Penicillium spp. while morphological changes were strain dependent at the concentrations tested. Diacetyl inhibited six out of six Penicillium spp. strains and increased spore size and number of germination sites in two out of six of the strains prior to germination.