<jats:title>Abstract</jats:title><jats:p>The fungus-growing ant-microbe symbiosis is an ideal system to study chemistry-based microbial interactions due to the wealth of microbial interactions described, and the lack of information on the molecules involved therein. In this study, we employed a combination of MALDI imaging mass spectrometry (MALDI-IMS) and MS/MS molecular networking to study chemistry-based microbial interactions in this system. MALDI IMS was used to visualize the distribution of antimicrobials at the inhibition zone between bacteria associated to the ant <jats:italic>Acromyrmex echinatior</jats:italic> and the fungal pathogen <jats:italic>Escovopsis</jats:italic> sp. MS/MS molecular networking was used for the dereplication of compounds found at the inhibition zones. We identified the antibiotics actinomycins D, X2 and X<jats:sub>0β</jats:sub>, produced by the bacterium <jats:italic>Streptomyces</jats:italic> CBR38; and the macrolides elaiophylin, efomycin A and efomycin G, produced by the bacterium <jats:italic>Streptomyces</jats:italic> CBR53.These metabolites were found at the inhibition zones using MALDI IMS and were identified using MS/MS molecular networking. Additionally, three shearinines D, F, and J produced by the fungal pathogen <jats:italic>Escovopsis</jats:italic> TZ49 were detected. This is the first report of elaiophylins, actinomycin X<jats:sub>0β</jats:sub> and shearinines in the fungus-growing ant symbiotic system. These results suggest a secondary prophylactic use of these antibiotics by <jats:italic>A</jats:italic>. <jats:italic>echinatior</jats:italic> because of their permanent production by the bacteria.