Peptides present an attractive scaffold for the development of new anticancer lead agents due to their accessibility and ease of modification. Synthetic ultrashort cationic lipopeptides, with four amino acids or less conjugated to a fatty acid, were developed to retain the biological activity of longer peptides in a smaller molecular size. Herein, we report the activity of amphiphilic lipotripeptides, lipotripeptoids and lipotetrapeptides against breast (MDA-MB-231, JIMT-1), prostate (DU145) and pancreas (MiaPaCa2) epithelial cancer cell lines. The lipotripeptide C16-KKK-NH2 and lipotetrapeptide C16-PCatPHexPHexPCat-NH2 were identified to possess anticancer activity. The latter lipotetrapeptide possess a short polyproline scaffold consisting of only two L-4R-aminoproline (PCat) and two L-4R-hexyloxyproline (PHex). However, all the prepared lipotripeptoids lack anticancer activity. The amphiphilic C16-PCatPHexPHexPCat-NH2 exhibited similar anticancer potency to the surfactant benzethonium chloride while superior activity was observed in comparison to myristylamine. Mechanistic studies revealed that the peptides do not lyse ovine erythrocytes nor epithelial cancer cells, thus ruling out necrosis as the mechanism of cell death. Surprisingly, the two lipopeptides exhibit different mechanisms of action that result in cancer cell death. The lipotripeptide C16-KKK-NH2 was found to induce caspase-mediated apoptosis while C16-PCatPHexPHexPCat-NH2 kills tumor cells independent of caspases.