Targeting cancer cells with a certain photosensitizer (PS) excited by appropriate laser irradiation to release singlet oxygen as a photodynamic therapy (PDT) remains a challengeable technique to destroy them effectively. This research aimed to assess the cytogenetic potential of 5-aminolevulinic acid (5-ALA) activated with laser irradiation (5-ALA/PDT) to damage the intact DNA of adenocarcinoma breast cancer cell line (MCF-7) and hepatocellular carcinoma cell line (HepG2). Cancer cells were treated with 0.5 and 1 mM 5-ALA for 4 h, the precursor of the photochemical protoporphyrin IX (PpIX), and then exposed to laser irradiation at 633 nm and 0.25W for 4 min before incubation for 24 h. Cytotoxicity of cancer cells was assessed using trypan blue exclusion assay. Genotoxicity was recorded by micronucleus test and comet assay. Both 5-ALA and laser irradiation, separately, were nontoxic on cancer cell lines, however, 5-ALA/PDT enhanced cell death in a concentration-dependent manner. Also, 5-ALA/PDT generated high percentages of micronuclei in MCF-7 and HepG2 cell lines as recorded in binucleated cells. Similarly, the mean percentages of DNA damage and tail moment ratio were intensified extremely in cancer cell lines treated with 5-ALA/PDT rather than non-treated cells or cells treated by 5-ALA or laser irradiation separately. In conclusion, the singlet oxygen of 5-ALA targets DNA of cancer cells when activated by laser irradiation. Therefore, photodynamic therapy is a perfect applicable process to damage DNA effectively during M-phase and prohibit cancer cells proliferation.