Quinone outside inhibitor (QoI) fungicide resistance in A. alternata populations was reported in Brazil for the first time in 2019, in São Paulo orchards, and the mutation G143A in cytochrome b ( cytb) was found in resistant isolates. Our study investigated the infectious process, production of Reactive Oxygen Species (ROS) and fungal cell death in resistant (QoI-R) and sensitive (QoI-S) Alternaria alternata pathotype tangerine (Aapt) isolates. Morphological characterization of Aapt isolates was performed using Confocal Laser Scanning Microscopy (CLSM). Alternaria brown spot (ABS) symptoms were produced by Aapt isolates on tangelo cv. BRS Piemonte. Germination of QoI-R conidia and production of germ tubes on tangelo leaflets treated with 100µg mL ^-1 of pyraclostrobin 18 hours after inoculation (hai) was observed using Scanning Electron Microscopy (SEM). At the same time, QoI-S conidial germination was inhibited on tangelo leaflets treated with pyraclostrobin. ROS production and cell death in Aapt isolates at high fungicide concentration were observed using CLSM. QoI-S conidia exhibited high ROS production, indicating high oxidative stress. When dyed with Propidium Iodate (PI), QoI-S conidia emitted red fluorescence, showing cell death and confirming their sensitive phenotype. In contrast, QoI-R conidia neither produced ROS nor exhibited red fluorescence, indicating no cell death and confirming their resistant phenotype. Therefore, our findings evidence that microscopic techniques may help characterize events during fungi-plant interactions, ROS production, cell death, and Aapt phenotypes resistant and sensitive to QoIs using fluorometric protocols. Research Highlights Our findings demonstrate the infection process, reactive oxygen species production, and cell death of Aapt isolates resistant and sensitive to quinone outside inhibitors (QoIs). Confocal laser scanning microscopy may help to characterize phenotypes resistant and sensitive to QoIs using fluorometric protocols.