Bacterial-fungal interactions significantly influence microbial community performance and elicit specific microbial behaviors, including the production of specialized metabolites. This study investigates bacterial adaptation to the presence of a fungus using a co-culture experimental evolution approach with Bacillus subtilis and Aspergillus niger. The results show that B. subtilis evolved to produce more surfactin, a lipopeptide, and enhance surface spreading, which inhibits fungal growth and acidification of the environment. These traits are linked to mutations in the DegS-DegU two-component system. The presence of surfactin causes bulging in fungal hyphae and cell wall stress, suggesting that the fungus selects for increased surfactin production, which inhibits fungal growth and enhances bacterial competitiveness. Bacteria and fungi share diverse habitats and interact in various ways, from mutualism to inhibition. These interactions influence microbial community structure and ecology, as well as the development and evolution of the interacting species. Bacteria and fungi can indirectly affect each other through diffusible signals like chemoattractants and quorum-sensing molecules. However, some interactions require close proximity or direct contact. The study highlights that B. subtilis evolved to produce more surfactin and spread more effectively in the presence of A. niger, which limits fungal expansion and acidification. This adaptation is driven by mutations in the DegS-DegU system, which regulate surfactin production and surface spreading. The study also shows that surfactin causes mislocalization of secretory vesicles in A. niger, leading to cell wall stress. The results suggest that the presence of the fungus selects for increased surfactin production, which inhibits fungal growth and facilitates bacterial competition. The findings demonstrate the potential of combining co-culture and laboratory evolution experiments to deepen understanding of bacterial-fungal interactions (BFls). Such co-culture adaptation methods could offer a general, genetically modified organism-free approach to enhance antifungal activities of biocontrol bacteria against pathogenic fungi. The study provides insights into the genetic and phenotypic evolution of B. subtilis in the presence of a fungus, highlighting the importance of surface colonization and competition in bacterial adaptation to fungal environments.Bacterial-fungal interactions significantly influence microbial community performance and elicit specific microbial behaviors, including the production of specialized metabolites. This study investigates bacterial adaptation to the presence of a fungus using a co-culture experimental evolution approach with Bacillus subtilis and Aspergillus niger. The results show that B. subtilis evolved to produce more surfactin, a lipopeptide, and enhance surface spreading, which inhibits fungal growth and acidification of the environment. These traits are linked to mutations in the DegS-DegU two-component system. The presence of surfactin causes bulging in fungal hyphae and cell wall stress, suggesting that the fungus selects for increased surfactin production, which inhibits fungal growth and enhances bacterial competitiveness. Bacteria and fungi share diverse habitats and interact in various ways, from mutualism to inhibition. These interactions influence microbial community structure and ecology, as well as the development and evolution of the interacting species. Bacteria and fungi can indirectly affect each other through diffusible signals like chemoattractants and quorum-sensing molecules. However, some interactions require close proximity or direct contact. The study highlights that B. subtilis evolved to produce more surfactin and spread more effectively in the presence of A. niger, which limits fungal expansion and acidification. This adaptation is driven by mutations in the DegS-DegU system, which regulate surfactin production and surface spreading. The study also shows that surfactin causes mislocalization of secretory vesicles in A. niger, leading to cell wall stress. The results suggest that the presence of the fungus selects for increased surfactin production, which inhibits fungal growth and facilitates bacterial competition. The findings demonstrate the potential of combining co-culture and laboratory evolution experiments to deepen understanding of bacterial-fungal interactions (BFls). Such co-culture adaptation methods could offer a general, genetically modified organism-free approach to enhance antifungal activities of biocontrol bacteria against pathogenic fungi. The study provides insights into the genetic and phenotypic evolution of B. subtilis in the presence of a fungus, highlighting the importance of surface colonization and competition in bacterial adaptation to fungal environments.