<p>Pineapple disease, caused by species of <i>Thielaviopsis</i>, is an important phytosanitary constraint associated with sugarcane planting material, leading to economic losses. In Brazil, its management relies predominantly on chemical control, with only one registered strain of <i>Trichoderma harzianum</i> available for biological control, underscoring the need to identify new biocontrol alternatives. This study aimed to develop and validate an <i>in vitro</i> method, based on sugarcane minisetts in a Gerbox system, for the assessment of pineapple disease, as well as to demonstrate its applicability for screening potential biological control agents. The phytopathogen was identified based on morphological characterization and partial sequencing of the <i>tef1-α</i> gene, and the isolate was determined to be <i>Thielaviopsis ethacetica</i>. Treatments consisted of five strains of <i>T. afroharzianum</i>, one commercial product based on <i>T. harzianum</i>, and controls with and without pathogen inoculation. Conidial suspensions were adjusted to 10⁷ conidia mL⁻¹ (<i>Trichoderma</i>) and 10⁵ conidia mL⁻¹ (<i>Thielaviopsis</i>). Surface-disinfested minisetts were microbiolized with antagonist suspensions, inoculated with the pathogen, placed in Gerbox, and incubated at 28&#xa0;°C for 21&#xa0;days. The proposed method allowed consistent differentiation among treatments, with <i>T. afroharzianum</i> strains showing significantly greater disease reduction (up to 93.06%) compared to the <i>T. harzianum</i> treatment and the inoculated control. Overall, the Gerbox-based minisett method proved to be a reproducible and effective approach for <i>in vitro</i> assessment of pineapple disease, with potential application in the preliminary screening of biological control agents.</p>