Melon is a major crop cultivated in the Brazilian Semiarid region. Several soilborne fungal pathogens cause root rot and vine decline (RRVD) in commercial production areas. This study evaluated the pathogenicity of Macrophomina pseudophaseolina isolates in melon accessions and characterised their differentially expressed protein profiles using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Two isolates differing in aggressiveness (CMM4807 and CMM4826) were inoculated into the moderately resistant accession C-14 and the susceptible accession MR-1 using the toothpick method. Protein extraction from stem and root tissues was performed using the phenol/SDS precipitation method, and quantified using the Bradford assay. Protein samples were analysed by SDS-PAGE. Root rot symptoms were observed in all inoculated melon seedlings, with the C-14 accession exhibiting moderate resistance to M. pseudophaseolina. Fewer differentially expressed protein bands were detected in the stem and root tissues of the C-14 accession when inoculated with the more aggressive isolate CMM4826. Accession C-14 exhibited moderate resistance to M. pseudophaseolina, a species associated with RRVD in melons. Protein analysis revealed differentially expressed bands after inoculation, with the number of bands influenced by the aggressiveness of the isolates, although both isolates were pathogenic. Further studies are necessary to identify these differentially expressed proteins and elucidate their functions in this plant-pathogen interaction.