Fungal and oomycete diseases are responsible for significant economic losses of cacao crops worldwide. Such diseases are caused mainly by the fungus Moniliophthora perniciosa, and the oomycetes Phytophthora citrophtora, P. capsici, and P. palmivora. The endophytic bacterium Burkholderia seminalis has showed antagonistic action against several plant pathogens. Herein, desorption electrospray ionization mass spectrometry imaging (DESI-MSI) was applied to map spatial metabolic distributions from B. seminalis and cacao pathogen monocultures, as well as to monitor interkingdom interactions of the endophytic bacterium with the fungus and oomycetes. DESI-MSI was successful in the direct analysis of fungus and oomycete pathogens on dehydrated agar plate, detecting several phospholipids and other unidentified metabolites. Chemical defense of B. seminalis against cacao pathogens was evidenced by the detection of partially diffuse rhamnolipid, which probably act in synergism with other unidentified diffuse metabolites.