Flooding caused or exacerbated by climate change has threatened plant growth and food production worldwide. The lack of knowledge on how crops respond and adapt to flooding stress imposes a major barrier to enhancing their productivity. Hence, understanding the flooding-responsive mechanisms of crops is indispensable for developing new flooding-tolerant varieties. Here, we examined the banana (Musa acuminata cv. Berangan) responses to soil waterlogging for 1, 3, 5, 7, 14, and 24 days. After waterlogging stress, banana root samples were analyzed for their molecular and biochemical changes. We found that waterlogging treatment induced the formation of adventitious roots and aerenchyma with conspicuous gas spaces. In addition, the antioxidant activities, hydrogen peroxide, and malondialdehyde contents of the waterlogged bananas increased in response to waterlogging stress. To assess the initial response of bananas toward waterlogging stress, we analyzed the transcriptome changes of banana roots. A total of 3508 unigenes were differentially expressed under 1-day waterlogging conditions. These unigenes comprise abiotic stress-related transcription factors, such as ethylene response factors, basic helix-loop-helix, myeloblastosis, plant signal transduction, and carbohydrate metabolisms. The findings of the study provide insight into the complex molecular events of bananas in response to waterlogging stress, which could later help develop waterlogging resilient crops for the future climate.