<jats:title>ABSTRACT</jats:title> <jats:p> Small-molecule signaling is one major mode of communication within the polymicrobial consortium of soil and rhizosphere. While microbial secondary metabolite (SM) production and responses of individual species have been studied extensively, little is known about potentially conserved roles of SM signals in multilayered symbiotic or antagonistic relationships. Here, we characterize the SM-mediated interaction between the plant-pathogenic bacterium <jats:named-content content-type="genus-species">Ralstonia solanacearum</jats:named-content> and the two plant-pathogenic fungi <jats:named-content content-type="genus-species">Fusarium fujikuroi</jats:named-content> and <jats:named-content content-type="genus-species">Botrytis cinerea</jats:named-content> . We show that cellular differentiation and SM biosynthesis in <jats:named-content content-type="genus-species">F. fujikuroi</jats:named-content> are induced by the bacterially produced lipopeptide ralsolamycin (synonym ralstonin A). In particular, fungal bikaverin production is induced and preferentially accumulates in fungal survival spores (chlamydospores) only when exposed to supernatants of ralsolamycin-producing strains of <jats:named-content content-type="genus-species">R. solanacearum</jats:named-content> . Although inactivation of bikaverin biosynthesis moderately increases chlamydospore invasion by <jats:named-content content-type="genus-species">R. solanacearum</jats:named-content> , we show that other metabolites such as beauvericin are also induced by ralsolamycin and contribute to suppression of <jats:named-content content-type="genus-species">R. solanacearum</jats:named-content> growth <jats:italic>in vitro</jats:italic> . Based on our findings that bikaverin antagonizes <jats:named-content content-type="genus-species">R. solanacearum</jats:named-content> and that ralsolamycin induces bikaverin biosynthesis in <jats:named-content content-type="genus-species">F. fujikuroi</jats:named-content> , we asked whether other bikaverin-producing fungi show similar responses to ralsolamycin. Examining a strain of <jats:named-content content-type="genus-species">B. cinerea</jats:named-content> that horizontally acquired the bikaverin gene cluster from <jats:italic>Fusarium</jats:italic> , we found that ralsolamycin induced bikaverin biosynthesis in this fungus. Our results suggest that conservation of microbial SM responses across distantly related fungi may arise from horizontal transfer of protective gene clusters that are activated by conserved regulatory cues, e.g., a bacterial lipopeptide, providing consistent fitness advantages in dynamic polymicrobial networks. <jats:p> <jats:bold>IMPORTANCE</jats:bold> Bacteria and fungi are ubiquitous neighbors in many environments, including the rhizosphere. Many of these organisms are notorious as economically devastating plant pathogens, but little is known about how they communicate chemically with each other. Here, we uncover a conserved antagonistic communication between the widespread bacterial wilt pathogen <jats:named-content content-type="genus-species">Ralstonia solanacearum</jats:named-content> and plant-pathogenic fungi from disparate genera, <jats:italic>Fusarium</jats:italic> and <jats:italic>Botrytis</jats:italic> . Exposure of <jats:named-content content-type="genus-species">Fusarium fujikuroi</jats:named-content> to the bacterial lipopeptide ralsolamycin resulted in production of the antibacterial metabolite bikaverin specifically in fungal tissues invaded by <jats:italic>Ralstonia</jats:italic> . Remarkably, ralsolamycin induction of bikaverin was conserved in a <jats:named-content content-type="genus-species">Botrytis cinerea</jats:named-content> isolate carrying a horizontally transferred bikaverin gene cluster. These results indicate that horizontally transferred gene clusters may carry regulatory prompts that contribute to conserved fitness functions in polymicrobial environments.