The potential as anticancer agents of 1,4-dihydropyridines (1,4-DHPs) and their pioneering urea derivatives have been evaluated in HeLa (cervix), Jurkat (leukaemia) and A549 (lung) cancer cell lines as well as on healthy mice. 1,4-DHPs show moderate cytotoxicity. However, when the urea moiety is introduced, an extraordinary increase in their antiproliferative activity is observed, proving an interesting synergy between these two scaffolds. Remarkably, when enantiomerically enriched samples are examined, they result to be in almost all cases less to equally active. This effect could be caused by a complex amalgam of physical and chemical contributions. The studied compounds present luminescent properties and a biodistribution study in cancer cells has been performed. Fluorescence microscopy showed that some of the 1,4-DHP derivatives accumulated in the lysosomes, whilst their urea counterparts targeted the cell membrane, which can be key to explain the different cytotoxic activity and imply a different mechanism of action. Finally, a preliminary in vivo study regarding the acute toxicity of some of these compounds on healthy mice has been conducted, using a concentration up to 7200 times higher than the corresponding IC₅₀ value. No downgrade in the welfare of the test subjects was observed, which could support their use in preclinical tumour models. Recently, we have been exploring the biological properties of 1-benzamido-1,4-dihydropyridine derivatives and the preliminary results on cytotoxicity will be commented.