High-dose chemotherapy combined with autologous transplantation using bone marrow or peripheral blood-derived stem cells (PBSC) is now widely used in the treatment of hematologic malignancies as well as some solid tumors like breast cancer (BC). However, some controversial results were recently obtained in the latter case. The presence of malignant cells in the autograft has been associated with the recurrence of the disease, and purging procedures are needed to eliminate this risk. The aim of this study was to evaluate the potential of the photosensitizer 4,5-dibromorhodamine methyl ester (TH9402), a dibrominated rhodamine derivative, to eradicate multiple myeloma (MM) and BC cell lines, while sparing more than 50% of normal pluripotential blood stem cells from healthy volunteers. The human BC MCF-7 and T-47D and MM RPMI 8226 and NCI-H929 cell lines were used to optimize the photodynamic purging process. Cell concentration and the cell suspension thickness as well as the dye and light doses were varied in order to eventually treat 1–2 L of apheresis. The light source consisted of two fluorescent scanning tubes emitting green light centered about 515 nm. The cellular uptake of TH9402 was measured during the incubation and washout periods and after photodynamic treatment (PDT) using spectrofluorometric analysis. The limiting dilution assay showed that an eradication rate of more than 5 logs is obtained when using a 40 min incubation with 5–10 μM dye followed by a 90 min washout period and a light dose of 5–10 J/cm² (2.8 mW/cm²) in all cell lines. Agitating the 2 cm thick cell suspension containing 20 × 10⁶ cells/mL during PDT was essential for maximal photoinactivation. Experiments on mobilized PBSC obtained from healthy volunteers showed that even more drastic purging conditions than those found optimal for maximal eradication of the malignant cell lines were compatible with a good recovery of hematopoietic progenitors cells. The absence of significant toxicity towards normal hematopoietic stem cells, combined with the 5 logs eradication of cancer cell lines induced by this procedure suggests that TH9402 offers an excellent potential as an ex vivo photodynamic purging agent for autologous transplantation in MM and BC treatment.