Penicillium spp. are considered a major spoilage fungus in dairy products. Due to the growing concerns over food safety issues and the demand for “clean label” food products from consumers, the use of lactic acid bacteria (LAB) as a bioprotective tool to control fungal spoilage of dairy products appears to be a promising alternative. Here, the antifungal activities of ten LAB cultures against five dairy-spoilage-associated Penicillium strains were studied in a model system, and the most potent bioprotective cultures were further tested in yoghurt. Lacticaseibacillus rhamnosus (L. rhamnosus) LRH01 and Lactiplantibacillus plantarum (L. plantarum) LP01 exhibited potent antifungal efficacy at low concentrations. The inhibitory effects of cell-containing fermentates (C-fermentates), cell-free fermentates (CF-fermentates), and volatiles produced by the two cultures were tested in a yoghurt serum medium. The C-fermentates showed antifungal effects, while the removal of cells from C-fermentates led to decreased antifungal activities. Volatiles alone displayed some antifungal efficiency, but less than the fermentates. In a yoghurt matrix, the specific effect of manganese depletion by the bioprotective cultures on mold growth was investigated. Here, the LAB cultures could completely suppress the growth of molds, while addition of manganese partially or fully restored the mold growth, demonstrating that manganese depletion played a key role in the antifungal activity of the tested LAB cultures in the yoghurt matrix. Both L. plantarum LP01 and L. rhamnosus LRH01 showed efficient antifungal activities in the yoghurt serum, while L. rhamnosus LRH01 exhibited the most potent inhibitory effects on Penicillium strains when added during the processing of the yoghurt with subsequent storage at 7 °C for 22 days. Our findings suggested that L. rhamnosus LRH01 could be a promising bioprotective culture for yoghurt biopreservation.