Rhabdomyosarcoma is a highly aggressive soft tissue cancer that predominantly affects children and adolescents. Current treatment outcomes are poor, highlighting the urgent need for potent therapeutic alternatives. Preclinical research on photodynamic therapy (PDT) continues to gain attention as a promising and minimally invasive treatment strategy. Recently, PDT using the heavy-atom-free photosensitizer dibenzothioxanthene imide (DBI), which targets cancer-associated G-quadruplex (G4) DNA, has demonstrated high efficacy at nanomolar concentrations. In here, transgenic zebrafish with rhabdomyosarcoma tumors were utilized to evaluate the therapeutic potential of DBI treatment. We demonstrate that photoactivated DBI efficiently induce localized tumor necrosis, resulting in significant rhabdomyosarcoma regression compared to untreated controls. In fact, in comparison to the healthy cells surrounding the tumor, a high level of G4s was detected, as visualized by a G4-specific antibody. Notably, muscle and nerve cells within the treated tumor area were particularly affected, further underscoring its potency. These findings position DBI as a promising candidate for PDT in the treatment of rhabdomyosarcoma, offering selective G4-targeting capabilities and delivering robust therapeutic outcomes in in vivo models.