<p>Bacteria form consortia as integral components of diverse ecosystems, where they interact with various organisms. Within these communities, bacterial–bacterial communication plays a pivotal role by driving numerous specific interactions. A key aspect of this chemical communication is the production of secondary metabolites. Recent research demonstrates that interspecies interactions between microorganisms can serve as physiological triggers, activating silent biosynthetic gene clusters and leading to the synthesis of novel secondary metabolites by the interacting species. This review focuses on mixed cultivation strategies involving actinobacteria, with an emphasis on utilizing mycolic acid-containing bacteria such as <i>Tsukamurella pulmonis</i> as inducer organisms. It comprehensively examines recent advances striving to understand these bacterial interactions, specifically involving the ability of actinomycetes to recognize and respond to mycolic acid-containing bacteria to activate secondary metabolism. Furthermore, the genetic basis of secondary metabolism activation was explored and newly discovered secondary metabolites induced by actinobacteria–mycolic acid-containing bacteria co-culture were highlighted. Finally, the integration of combined-culture strategies with genetic engineering methods and the ecological relevance of actinobacteria–mycolic acid-containing bacteria interactions were discussed. These bacterial interactions provide an excellent model system for understanding the molecular mechanisms regulating secondary metabolism and could open new avenues for drug discovery.</p>