Biosurfactants are amphiphilic molecules with surface tension reducing activities. Among biosurfactant producers, fungi have been identified as promising organisms. While many studies have investigated biosurfactant production in fungal species from the Ascomycota and Basidiomycota phyla, less is known concerning species from the Mucoromycota phylum. In this context, the aim of this study was to screen and optimize biosurfactant production in 24 fungal strains, including seven Mucor, three Lichtheimia, and one Absidia species. After cultivation in a medium stimulating surfactant production, the surface activity of cell-free supernatants was measured using both oil spreading and parafilm M tests. Among them, five Mucor strain cell-free supernatants belonging to M. circinelloides, M. lanceolatus, M. mucedo, M. racemosus, and M. plumbeus, showed oil repulsion. Then, the impact of the medium composition on surfactant production was evaluated for eight strains. Three of them, i.e., Mucor circinelloides UBOCC-A-109190, Mucor plumbeus UBOCC-A-111133, and Mucor mucedo UBOCC-A-101353 showed an interesting surfactant production potential, reducing the medium surface tension to 36, 31, and 32 mN/m, respectively. A preliminary characterization of the surfactant molecules produced by these strains was performed and showed that these compounds belonged to the glycolipid family.