Propolis is a natural product widely known for its medicinal properties. In this work, fungi present on propolis samples were isolated, identified and tested for the production of antimicrobial metabolites. Twenty-two fungal isolates were obtained, some of which were identified as Alternaria alternata, Aspergillus flavus, Bipolaris hawaiiensis, Fusarium merismoides, Lasiodiplodia theobromae, Penicillium citrinum, Penicillium crustosum, Penicillium janthinellum, Penicillium purpurogenum, Pestalotiopsis palustris, Tetracoccosporium paxianum and Trichoderma koningii. These fungi were grown in liquid media to obtain crude extracts that were evaluated for their antibiotic activity against pathogenic bacteria, yeast and Cladosporium cladosporioides and A. flavus. The most active extract was obtained from L. theobromae (minimum inhibitory concentration = 64 μg/mL against Listeria monocitogenes). Some extracts showed to be more active than the positive control in the inhibition of Staphylococcus aureus and L. monocitogenes. Therefore, propolis is a promising source of fungi, which produces active agents against relevant food poisoning bacteria and crop-associated fungi.