Ewing sarcomas (ES) are aggressive primary bone tumors that require radical therapy. Promising low toxicity, 5-aminolevulinic acid (5-ALA)-mediated photodynamic therapy (PDT) could enhance the effectiveness of conventional treatment modalities (e.g., doxorubicin (DOX)), improving, thus, the anti-tumorigenic effects. In this study, we investigated the effects of DOX and 5-ALA PDT alone or in combination on three different human ES cell lines. Cell viability, reactive oxygen species (ROS) production, and cellular stiffness were measured 24 h after PDT (blue light-wavelength 436 nm with 5-ALA) with or without DOX. ES cell lines have a different sensitivity to the same doses and exposure of 5-ALA PDT. DOX in combination with 5-ALA PDT was found to be effective in impairing the viability of all ES cells while also increasing cytotoxic activity by high ROS production. The stiffness of the ES cells increased significantly (p < 0.05) post treatment. Overall, our results showed that across multiple ES cell lines, 5-ALA PDT can successfully and safely be combined with DOX to potentiate the therapeutic effect. The 5-ALA PDT has the potential to be a highly effective treatment when used alone or in conjunction with other treatments. More research is needed to assess the effectiveness of 5-ALA PDT in in vivo settings.