Esophageal squamous cell carcinoma (ESCC) is a highly prevalent malignancy worldwide. Moreover, ESCC remains poorly characterized at the molecular level, which contributes to limited therapeutic options and an overall poor prognosis. In this context, HMGA family members, which are overexpressed in tumors but almost absent in healthy adult tissues, seem to represent promising therapeutic targets. These proteins act by binding to AT-hook DNA-binding motifs and may regulate the expression of several genes associated with tumor progression. Therefore, integrating in silico, translational, and in vitro approaches, we investigated the functional consequences of blocking HMGA2–DNA interaction in ESCC tumor progression by using netropsin, a site-specific ligand for AT-rich DNA regions. Our results demonstrate that netropsin treatment significantly reduced cell viability, migration, and cell cycle progression, thereby promoting apoptosis. Furthermore, netropsin treatment was capable of partially reverting Epithelial–Mesenchymal Transition (EMT) activation associated with HMGA2 expression, by downregulating EMT activators, such as Slug and Twist. Finally, the netropsin treatment sensitizes ESCC cells to chemotherapeutic treatment with 5-Fluorouracil. Taken together, our findings highlight that AT binding-specific blockade could be correlated with the inhibition of HMGA2 and may reveal a promising approach to better understand ESCC progression.