Background and Objectives: Fireweed (Chamerion angustifolium L.) possesses antibacterial, antioxidant, anti-inflammatory, and anti-cancer properties. This study evaluated the effects of aqueous fireweed leaf extracts and their major compound, oenothein B, on the viability and mitochondrial function of Caco-2 colon cancer cells, emphasizing the impact of leaf fermentation. Materials and Methods: Cells were treated for 48 h with oenothein B and aqueous extracts from unfermented (NF) and fermented leaves (F 24 h, F 48 h). Cell viability and mitochondrial function were assessed by MTT assay and high–resolution respirometry. Results: IC₅₀ values were 0.843 mg/mL (NF), 1.548 mg/mL (F 24 h), 1.931 mg/mL (F 48 h), and 0.09 mg/mL (57 µM) (oenothein B). Mitochondrial respiration decreased in up to 67% (glutamate/malate) and 61% (succinate) in both fermented and unfermented groups, while oenothein B increased in leak respiration by 34–73% but reduced oxidative respiration by 24%. Conclusions: Aqueous extracts of fireweed from both unfermented and fermented leaves significantly reduced Caco-2 cell viability and mitochondrial function. Oenothein B on its own had a stronger effect on cell viability, but a weaker effect on mitochondrial function, compared to fireweed extracts.