Crotalicidin (Ctn) and its fragment Ctn[15-34] are snake-venom-derived, cathelicidin-related peptides outstanding for their promising antimicrobial, antifungal, and antitumoral properties. In this study, we describe their membranolytic mechanisms as well as their putative interference with intracellular targets, both contributing to their antitumoral action against a pro-monocytic leukemia cell line. Initial flow cytometry assays demonstrated peptide ability to induce tumor cell membrane permeabilization and caspase-dependent apoptosis, without total activity reduction by serum proteases up to 24 h (Ctn) and 18 h (Ctn[15-34]). In addition, both Ctn and Ctn[15-34] showed preference for tumor cells rather than healthy cells, with selectivity ratios (tumoral vs healthy cells) of 17 and 7, respectively. Further microscopy and flow cytometry studies suggested their preferential accumulation in the cytoplasmic membrane and nucleus and proposed multiple predominant routes of peptide uptake, including direct entry and endocytosis. Affinity purification followed by proteomic identification experiments revealed both peptides to interact with proteins involved in DNA and protein metabolism, cell cycles, signal transduction, and/or programmed cell death, among others. These results suggest a putative role of Ctn and Ctn[15-34] to interact with key intracellular pathways, ultimately contributing to tumor cell death by necrosis/apoptosis. Altogether, this work proposes a dual mechanism underlying the antitumoral activity of Ctn and Ctn[15-34] and reinforces their potential as future therapeutic drugs.