ABSTRACT Bacterial-fungal interactions (BFI) are critical drivers of ecosystem functions, and their molecular mechanisms remain incompletely understood. Chemical signals are crucial mediators of microbial communication, but the signaling mechanisms of terpene compounds secreted by fungi are not fully elucidated. In this study, the environmental bacterium Lysobacter enzymogenes YC36 ( Le YC36) was employed to systematically investigate the regulatory mechanism of α-terpinene, a fungal-derived terpene, on biofilm formation and antifungal activity. Our results indicate that α-terpinene enhances the competitiveness of Le YC36 in BFI by promoting the biosynthesis of antifungal effectors, heat-stable antifungal factor (HSAF), and fungal cell wall hydrolase GluB, as well as biofilm formation. Furthermore, we reveal the α-terpinene-mediated signaling pathway in Le YC36. Le YC36 detects the fungal signal α-terpinene through Le CzcS, the sensor histidine kinase of the two-component system Le CzcS/ Le CzcR, which then transmits the information to the regulator Le CzcR. The phosphorylated Le CzcR directly stimulates the expression of downstream functional genes Lepks/nrps HSAF ( Lehsaf ), LegluB , and Lepsl , which enhance the production of antifungal effectors (HSAF and GluB), improve biofilm formation, and ultimately boost the competitiveness of Le YC36 against fungi. This study elucidates the role of α-terpinene in BFI and its regulatory mechanisms, which provides a novel perspective on communication and cooperative adaptation among microorganisms. IMPORTANCE The complex interactions between bacteria and fungi play a crucial role in maintaining ecosystem balance and biodiversity. This study elucidates the molecular mechanism by which α-terpinene, as a fungal-derived signaling molecule, regulates biofilm formation and antifungal activity in Lysobacter enzymogenes YC36 ( Le YC36) and preliminarily establishes the signaling regulatory pathway of α-terpinene. These findings advance our understanding of interkingdom signals between bacteria and fungi and provide a theoretical framework for developing biocontrol technologies using microbial signaling molecules.