Introduction: Pancreatic ductal adenocarcinoma (PDAC) is the sixth leading cause of death worldwide. PDAC carries a 5-y survival of less than 10%, as it is often diagnosed at a late stage and is widely refractory to available therapies. PDAC tumors are hypoperfused, resulting in poor nutrient delivery. To exist in this hostile microenvironment, PDAC cells rely on intracellular and extracellular scavenging pathways to acquire metabolic substrates for growth. Autophagy and other lysosome-dependent recycling pathways are aberrantly regulated in PDAC. Although autophagy modulation is an emerging therapeutic strategy for PDAC, the co-dependences induced by lysosomal inhibition have not been systematically explored. Cationic amphiphilic drugs (CADs) accumulate in lysosomes, destabilize their membranes, and can have anti-tumorigenic effects. CADs include hundreds of pharmacologic agents used to treat a broad spectrum of common diseases. The aim of this study was to investigate anti-PDAC activity of several clinically-approved and newly synthesized CADs. Material and methods: Imidazoline, quinoline, and chrysene derivatives were examined. Anti-tumor activity of CADs was evaluated in human PDAC cell lines in vitro and in Tg(fli1:EGFP) zebrafish model in vivo. Effects on mitochondria, lysosomes, and autophagy flux were examined by immunofluorescent microscopy. Levels of reactive oxygen species (ROS), changes in the mitochondrial membrane potential, and induction of apoptosis by the selected CADs were evaluated by flow cytometry. Results: The newly synthesized derivatives of quinoline and chrysene induced apoptosis in PDAC cells in vitro in the 2–10μM range, while the only FDA-approved imidazoline with apoptotic activity was rilmenidine, at doses higher than 100μM. Most of the tested compounds induced the expansion of the acidic compartment, as measured by acridine orange and LAMP1 staining, and modulated autophagy, as seen by LC3 staining intensity and distribution. Mitochondrial oxidative stress was induced by the tested CADs as well as the dissipation of the mitochondrial membrane potential (ΔΨm). Ultimately, both newly synthesized CADs and rilmenidine limited the growth and spread of PANC1 cancer cells in the Tg(fli1:EGFP) zebrafish model. Conclusion: Quinoline and chrysene CADs had the potential for a dual lysosome/mitochondrion targeting in PDAC cells and were inducing apoptosis almost in a nM range. While rilmenidine was less potent than newly synthetized CADs in inducing cell death in vitro, all the tested compounds had significant anti-tumor effects in the zebrafish model. These results imply that CADs have promising anti-PDAC effects and that effects on the other cells in the tumor microenvironment other than the cancer cells themselves have to be examined. No conflict of interest.