Background <p>Tumor hypoxia is a significant factor in cancer progression, metastasis, and therapy resistance, leading to poor patient outcomes. Hypoxia-activated prodrugs (HAPs) are a class of agents that selectively target these hypoxic environments. They remain inactive under normal oxygen conditions but are activated by low oxygen levels. TH-302 (evofosfamide), a nitroimidazole mustard, is a clinically advanced HAP. This study aimed to investigate the effects of TH-302 in three Ewing's sarcoma (ES) cell lines.</p> Methods <p>TH-302 was assessed for its effects on DNA damage, cell proliferation, cell death, mitochondrial depolarization, caspase-3/7 activation, and the emergence of sub-G1 cell populations using flow cytometry, fluorescence microscopy and quantitative real time (qRT)-PCR. These effects were compared under different oxygen concentrations.</p> Results <p>TH-302 induced DNA damage and reduced cell number in ES cells by over 60%, preferentially under hypoxic conditions, independent of the cellular p53 status. TH-302 caused cell death in up to 40% of cells and mitochondrial depolarization in up to 60% of cells. Additionally, TH-302-induced caspase-3/7 activation and increased sub-G1 cell populations predominantly under hypoxia, with the most pronounced effects occurring at 0.2% environmental oxygen compared to 1% O₂.</p> Conclusion <p>These in vitro findings suggest that TH-302 may be efficacious in ES. This provides a rationale for further in vivo investigations into the potential of TH-302 as a treatment for ES.</p>