A series of ten α , β -unsaturated benzotriazolyl-1,3,4-oxadiazole derivatives was synthesized and all compounds were evaluated in vitro against three breast cancer cell lines (MCF-7, MDA-MB-231 and 4T1) at different concentrations (0.1, 0.5, 1, 2, 3, 4 and 5 mg/mL). The results showed that compounds 6a, 6c, 6d, 6f, 6g, and 6i displayed acceptable anticancer activity, where compound 6f was the most active on the three cell lines (IC50 = 0.80, 0.07, and 0.30 mg/mL, respectively). Regarding the cytotoxicity assay, the compounds exhibited modest toxicity on murine splenocytes and peripheral human blood cells at the highest concentration tested (5 mg/mL). Compound 6f was further evaluated at different concentrations showing moderate cytotoxicity at the 5 mg/mL concentration and negligible cytotoxicity at the minimum concentration evaluated (0.05 mg/mL). Finally, the compounds 6a, 6c, 6d, 6f, 6g, 6i, and 6j were evaluated as fluorescence markers due to their ability to be internalized into MCF-7 cells.