Cancer incidence and mortalities are growing worldwide, therefore research and development of more effective and less invasive treatments, such as photodynamic therapy, are needed. Herein, we investigated the methylene blue (MB) photoactivation effects in lung epithelial cells (BEAS-2B) and lung adenocarcinoma cells (H-441). The reactive oxygen species (ROS) produced by the laser photoactivation of MB in aqueous solutions and cell cultures were measured with probes, and the cell viability was evaluated with a colorimetric assay. MB up to 31.26 μM did not induce detectable effects in BEAS-2B cells. However, H-441 cells presented adverse effects below that concentration in the same range of fluencies studied. These results are in concordance with the ROS production in H-441 cells, while in BEAS-2B cells the production of ROS was less significant compared to the control. Photoactivation of MB at concentrations below 31.26 μM could be used for the selective treatment of H-441 cells over non-cancer cells.