Fungi grow in competitive environments, and to cope, they have evolved strategies, such as the ability to produce a wide range of secondary metabolites. This begs two related questions. First, how do secondary metabolites influence fungal ecology and interspecific interactions? Second, can these interspecific interactions provide a way to "see" how fungi respond, chemically, within a competitive environment? To evaluate these, and to gain insight into the secondary metabolic arsenal fungi possess, we co-cultured , a genetically tractable fungus that produces a suite of mycotoxins, with , a fungus that produces the fungistatic compound and FDA-approved drug, griseofulvin. To monitor and characterize fungal chemistry , we used the droplet-liquid microjunction-surface sampling probe (droplet probe). The droplet probe makes a microextraction at defined locations on the surface of the co-culture, followed by analysis of the secondary metabolite profile via liquid chromatography-mass spectrometry. Using this, we mapped and compared the spatial profiles of secondary metabolites from both fungi in monoculture versus co-culture. predominantly biosynthesized griseofulvin and dechlorogriseofulvin in monoculture. In contrast, under co-culture conditions a deadlock was formed between the two fungi, and biosynthesized the same two secondary metabolites, along with dechloro-5'-hydroxygriseofulvin and 5'-hydroxygriseofulvin, all of which have fungistatic properties, as well as mycotoxins like cytochalasin D and cytochalasin C. In contrast, in co-culture, increased the production of the mycotoxins fumitremorgin B and verruculogen, but otherwise remained unchanged relative to its monoculture. To evaluate that secondary metabolites play an important role in defense and territory establishment, we co-cultured lacking the master regulator of secondary metabolism with . We found that the reduced secondary metabolite biosynthesis of the Δ strain of eliminated the organism's ability to compete in co-culture and led to its displacement by . These results demonstrate the potential of chemical analysis and deletion mutant approaches for shedding light on the ecological roles of secondary metabolites and how they influence fungal ecological strategies; co-culturing may also stimulate the biosynthesis of secondary metabolites that are not produced in monoculture in the laboratory.