Coffee corky-root disease causes serious damages to coffee crop and is linked to combined infection of Fusarium spp. and root-knot nematodes Meloidogyne spp. In this study, 70 Fusarium isolates were collected from both roots of healthy coffee plants and with corky-root disease symptoms. A phylogenetic analysis, and the detection of pathogenicity SIX genes and toxigenicity Fum genes was performed for 59 F. oxysporum and 11 F. solani isolates. Based on the molecular characterization, seven F. oxysporum and three F. solani isolates were assessed for their pathogenicity on coffee seedlings under optimal watering and water stress miming root-knot nematode effect on plants. Our results revealed that a drastic increment of plant colonization capacity and pathogenicity on coffee plants of some Fusarium isolates was caused by water stress. The pathogenicity on coffee of F. solani linked to coffee corky-root disease and the presence of SIX genes in this species were demonstrated for the first time. Our study provides evidence for understanding the pathogenic basis of F. oxysporum and F. solani isolates on coffee and revealed the presence of SIX and Fum genes as one of their pathogenicity-related mechanisms. We also highlight the relevance of chlorophyll, a fluorescence as an early and high-throughput phenotyping tool in Fusarium pathogenicity studies on coffee.