Cerenkov radiation (CR) can be used as an internal light source in photodynamic therapy (PDT). Methotrexate (MTX) and paclitaxel (PTX), chemotherapeutic agents with wide clinical use, have characteristics of photosensitizers (PS). This work evaluates the possibility of photoexciting MTX and PTX with CR from 18 F-FDG to produce reactive oxygen species (ROS) capable of inducing cytotoxicity. PTX did not produce ROS when excited by CR from 18 F-FDG, so it is not useful for PDT. In contrast, MTX produces 1 O 2 (detected by ABMA) in amounts sufficient to significantly decrease the viability of the T47D cells. MTX solutions of 100 nM combined with 18 F-FDG activities of 50 (1.85 MBq) and 100 μCi (3.7 MBq) produced a significant decrease in cell viability to (50.09 ± 4.95) and (47.96 ± 11.19)%, respectively, compared to MTX (66.29 ± 5.92)% and 18 F-FDG (91.35 ± 7.00% for 50 μCi and 99.43 ± 11.03% for 100 μCi) alone. Using the CellRox Green reagent, the intracellular production of ROS was confirmed as the main mechanism of cytotoxicity. The results confirm the therapeutic potential of photoactivation with CR and the synergy of the combined treatment with chemotherapy + photodynamic therapy (CMT + PDT). The combination of chemotherapeutic agents with PS properties and β-emitting radiopharmaceuticals, previously approved for clinical use, will make it possible to shorten the evaluation stages of new CMT + PDT systems.