Resumo. Mycelia from 3 isolates of Mycosphaerella fijiensis (Mak 01, Mak 02 and Kaw 10) were fragmented for 3 and 1 minutes, and tested at 5 mg mL-1, 10 mg mL-1 and 15 mg mL-1 for virulence in the screen house. Disease severity was found to depend on the level of fragmentation and concentration. Inoculation of Gros Michel plantlets with mycelia fragmented for 3 minutes resulted in significantly higher levels of disease severity (AUDPC) than fragmentation for 1 minute for all the isolates. For both times of fragmentation, AUDPC increased with concentration. The highest and lowest AUDPCs for both times of fragmentation were achieved with concentrations of 15 and 5 mg mL-1, respectively. Overall, all the three isolates demonstrated high levels of virulence, with Mak 01 causing the highest severity (AUDPC - 98.5) when compared to Mak 02 (AUDPC - 93.5) and Kaw 10 (AUDPC - 92.2) for 3-minute fragmentation and concentration of 15 mg mL-1. Collectively, our data demonstrate that a potent inoculum of Mycosphaerella fijiensis for in vitro studies can be reliably prepared by fragmenting weighed mycelia in a blender.