Basil (Ocimum sp.), a medicinal herb is used fresh and/or dry in various (culinary, cosmetic and pharmaceutical) preparations. Fusarium wilt caused by the fungus Fusarium oxysporum f. sp. basilici is limiting basil cultivation in many countries. Since the leaf is the edible part in basil, new approaches are required to identify, and to prevent the spread of Fusarium pathogens. Desorption electrospray ionization mass spectrometry (DESI MS) was used for imaging thin layer chromatography (TLC) - imprints of leaves of three different species of basil (Ocimum basilicum L., O. tenuiflorum L., and O. gratissimum L.), and the molecular manifestations during Fusarium contamination are recorded. DESI MS images showed the chemotaxonomic differences of basil species and the changes in metabolite ion peaks during pathogen infection. Besides easy detection of reported toxic metabolite(s) of the pathogen(s), the results include molecular images showing spatial distribution of all coexisting surface-bound metabolites in plant leaves, their fragment ions, and the transient changes in their spatial distribution during Fusarium attack under natural conditions. Demonstration of the same protocol to image seedling, young/mature leaves, basil/other related plant (Patchouli - Pogostemon cablin (Blanco) Benth.), wilt/other disease symptoms shows that prior knowledge of the metabolite profile of the plant/pathogen is not required. This rapid detection method can be tailored to large scale screening programs for plant diseases suggesting potential implications in agriculture and quarantine requirements. © The Royal Society of Chemistry.