Bioprospecting can uncover new yeast strains and species with valuable biotechnological and industrial traits, such as the capacity to convert different carbon sources in industrial side- and waste streams into bioproducts. In this study, we conducted untargeted bioprospecting in tropical West Africa, collecting 1996 isolates and determining their growth in 70 different environments. While the collection contains numerous isolates with the potential to assimilate several cost-effective and sustainable carbon and nitrogen sources, we focused on characterizing strains capable of growing on lactose, the main carbon source in the abundant side stream cheese whey from dairy industries. Our screening identified 30 different species capable of growing on lactose in both synthetic complete yeast medium and whey, with 20 of these also capable of growth in lactose-containing minimal medium. The lactose-assimilating yeast collection contains both ascomycetes and basidiomycetes, and includes species known to be lactose-assimilating, species not previously known to grow on lactose, and candidates for new species. Observed differences in growth profiles and ratios of intra- and extracellular lactase activities suggest that the yeasts use a range of different strategies to metabolize lactose. Notably, several basidiomycetes, including Apiotrichum mycotoxinivorans, Papiliotrema laurentii and Moesziomyces antarcticus, which accumulated up to 40% of their cell dry weight, proving that they can convert lactose into a bioproduct of significant biotechnology interest. Our findings highlight the value of bioprospecting of underexplored regions to discover yeast strains with substantial biotechnological potential, which may aid the transition to a circular bioeconomy.