Black Sigatoka, a devastating disease of bananas and plantains worldwide, is caused by the fungus Mycosphaerella fijiensis. Several banana cultivars such as 'Yangambi Km 5' and Calcutta IV, have been known to be resistant to the fungus, but the resistance has been broken in 'Yangambi Km 5' in Costa Rica. Since the resistance of this variety still persists in Mexico, the aim of this study was to compare the in vitro and in planta secretomes from two avirulent and virulent M. fijiensis isolates using proteomics and bioinformatics approaches. We aimed to identify differentially expressed proteins in fungal isolates that differ in pathogenicity and that might be responsible for breaking the resistance in 'Yangambi Km 5'. We were able to identify 90 protein spots in the secretomes of fungal isolates encoding 42 unique proteins and 35 differential spots between them. Proteins involved in carbohydrate transport and metabolism were more prevalent. Several proteases, pathogenicity-related, ROS detoxification and unknown proteins were also highly or specifically expressed by the virulent isolate in vitro or during in planta infection. An unknown protein representing a virulence factor candidate was also identified. These results demonstrated that the secretome reflects major differences between both M. fijiensis isolates.