Bacterial-fungal interactions (BFIs) are important in ecosystem dynamics, especially within the soil rhizosphere. The bacteriumBacillus velezensisSQR9 and the fungusTrichoderma guizhouenseNJAU 4742 have garnered considerable attention due to their roles in promoting plant growth and protecting their host against pathogens. In this study, we utilized these two model microorganisms to investigate BFI. We firstly demonstrate that while co-inoculation ofB. velezensisandT. guizhouensecould promote tomato growth, these two microorganisms display mutual antagonism on agar solidified medium. To resolve this contradiction, we developed an inoculation method, that allowsB. velezensiscolonization ofT. guizhouensehyphae and performed a transcriptome analysis. During colonization of the fungal hyphae,B. velezensisSQR9 upregulates expression of biofilm related genes (e.g.eps, tasA, andbslA)that is distinct from free-living cells. This result suggested an intricate association between extracellular matrix expression and hyphae colonization. In accordance, deletionepsD, tasA, orbothepsDandtasAgenes ofB. velezensisdiminished colonization of theT. guizhouensehyphae. The insights from our study demonstrate that soil BFIs are more complex than we understood, potentially involving both competition and cooperation. These intricate biofilm-mediated BFI dynamics might contribute to the remarkable diversity observed within soil microbiota, providing a fresh perspective for further exploration of BFIs in the plant rhizosphere.