Ceratocystis wilt of yerba mate (Ilex paraguariensis) caused by the fungus Ceratocystis fimbriata was observed in 2018 in commercial plantations located in São Mateus do Sul, PR and Arvorezinha, RS, Brazil, where mortality of the plants was up to 5%. Due to the need of control measures for this disease and to the lack of knowledge on the impact of this pathogen this new host, this study aimed to evaluate the aggressiveness of C. fimbriata isolates collected from yerba mate plantations and screen for host responses in yerba mate clones to Ceratocystis wilt. Two experiments were conducted in a completely randomized design, with 11 treatments (isolates) to assess aggressiveness of the isolates. Two experiments were conducted to assess resistance of yerba mate to C. fimbriata in a completely randomized design in a 6 × 2 factorial scheme (6 clones × 2 isolates) in two different environmental conditions (plant growth chamber and greenhouse). The isolates of C. fimbriata showed different levels of aggressiveness to yerba mate, and all evaluated clones were susceptible to Ceratocystis wilt. The plant growth chamber condition led to a faster death of the plants compared with greenhouse. This information will be useful to understand the pathosystem and seek strategies for controlling this disease in yerba mate.