Cancer is expected to rank as the leading cause of death and the most important barrier to increase life expectancy in the 21st century. Colorectal cancer (CRC) has been ranked as one of the most incident cancer types and one of the most mortal. Overall, the number of effective anti-cancer agents approved for use in humans is still very limited. Moreover, tumor resistance and secondary effects stemming from classical chemotherapy remain a major clinical problem, reinforcing the need for the development of novel drugs. In recent years, benzo[a]phenoxazines derivatives have been shown to possess anticancer activity, which has created interest in exploring the potential of these compounds as anticancer drugs. We have recently synthesized and evaluated the biological activity of an array of new benzo[a]phenoxazines and demonstrated that they display a varied antiproliferative activity against Saccharomyces cerevisiae. In the present study, we selected four of our most active compounds and evaluated their anticancer activity in Colorectal Cancer (CRC) cells. Our results showed that all compounds had a more toxic effect on CRC cell lines compared to non-tumor cell lines. We detected that the compounds accumulated on the lysosomes and induced lysosomal membrane permeabilization (LMP) that resulted in cytosolic acidification and apoptotic cell death in CRC cells. These observations highlight compounds of this class as promising candidates to be explored as new anticancer targeted agents for CRC treatment, using LMP as a novel cancer therapeutic approach.