Pokkah boeng disease (PBD), which is caused by Fusarium verticillioides, is a major sugarcane disease in Southeast Asian countries. Breeding varieties to become resistant to F. verticillioides is the most effective approach for minimizing the damage caused by PBD, and identifying genes mediating resistance to PBD via molecular techniques is essential. The production of reactive oxygen species (ROSs) is one of a cell’s first responses to pathogenic infections. Plant peroxisomes play roles in several metabolic processes involving ROSs. In this study, seedlings of YT94/128 and GT37 inoculated with F. verticillioides were used to identify PBD resistance genes. The cells showed a high degree of morphological variation, and the cell walls became increasingly degraded as the duration of the infection increased. There was significant variation in H₂O₂ accumulation over time. Catalase, superoxide dismutase, and peroxidase activities increased in both seedlings. Analysis of differentially expressed genes (DEGs) revealed that peroxidase-metabolism-related genes are mainly involved in matrix protein import and receptor recycling, adenine nucleotide transport, peroxisome division, ROS metabolism, and processes related to peroxisomal membrane proteins. The expression levels of SoCATA1 and SoSOD2A2 gradually decreased after sugarcane infection. F. verticillioides inhibited the expressions of C5YVR0 and C5Z4S4. Sugarcane infection by F. verticillioides disrupts the balance of intracellular ROSs and increases the cell membrane’s lipid peroxidation rate. Defense-related enzymes play a key regulatory role in maintaining a low, healthy level of ROSs. The results of this study enhance our understanding of the mechanism through which peroxisomes mediate the resistance of sugarcane to PBD and provide candidate genes that could be used to breed varieties with improved traits via molecular breeding.