The SARS-CoV-2-encoded 2-E channel is critical in the viral life cycle and pathogenesis. By facilitating viral replication, it promotes the dysregulation of inflammatory pathways, leading to cytokine storm, and triggers DNA damage response (DDR), thus exacerbating disease progression. The 2-E channel, a viroporin, is a promising antiviral target. However, the lack of specific inhibitors and effective screening methods has hindered therapeutic exploitation of the 2-E channel. To address this gap, we report on a fluorescence-based screening assay that targets the 2-E channel activity, resulting in the identification of potential inhibitory molecules. After performing both electrophysiological studies and surface plasmon resonance (SPR) analyses, we identified the top-ranked candidate, TPN10518, as a pore-blocking inhibitor of the 2-E channel. TPN10518 binds to a hydrophobic pocket in the C-terminal vestibule of the 2-E channel, thereby inhibiting its activity. Functional evaluation showed that TPN10518 exhibits significant antiviral efficacy in vitro, while, at the same time, effectively protecting against 2-E channel-mediated host damage and suppressing cytokine storm caused by dysregulated homeostasis of inflammatory pathways in vivo. Therefore, our work introduces a screening method for targeting 2-E channels, establishes the 2-E channel as a viable therapeutic target against SARS-CoV-2, and identifies TPN10518 as a promising antiviral candidate.