The ability of β-carboline alkaloids to effectively impair cell growth and induce cell death in diverse cancers highlights their potential as innovative leukemia treatments. This study aimed to evaluate the antineoplastic activities of 12 β-carboline derivatives, differing in substituents at the 1- and 3-positions of the β-carboline nucleus, against a panel of leukemia cell lines. We investigated the cytotoxic effects of these derivatives, focusing on their modulation of reactive oxygen species (ROS) generation, autophagy, and key signaling pathways. Our results demonstrated that β-carboline treatment induced potent, concentration-dependent antineoplastic activity, resulting in significant reductions in cell viability and increased cell death. Importantly, healthy donor mononuclear cells showed limited sensitivity to these compounds. Mechanistic studies revealed that the antineoplastic activity involved the inhibition of ROS production, which subsequently disrupted PI3K/AKT and MAPK/ERK signaling, and the modulation of autophagy processes. In vivo, a xenograft mouse model confirmed the efficacy of these derivatives, showing a substantial reduction in AML burden within both peripheral blood and bone marrow. These data suggest that β-carboline derivatives offer a targeted approach to malignant cell elimination, making them promising candidates for leukemia therapy by interfering with critical pathways that promote disease progression and drug resistance.