Cardiotoxins (CaTx) of the three-finger toxin family are one of the main components of cobra venoms. Depending on the structure of the N-terminal or the central polypeptide loop, they are classified into either group I and II or P- and S-types, respectively, and toxins of different groups or types interact with lipid membranes variably. While their main target in the organism is the cardiovascular system, there is no data on the effects of CaTxs from different groups or types on cardiomyocytes. To evaluate these effects, a fluorescence measurement of intracellular Ca2+concentration and an assessment of the rat cardiomyocytes’ shape were used. The obtained results showed that CaTxs of group I containing two adjacent proline residues in the N-terminal loop were less toxic to cardiomyocytes than group II toxins and that CaTxs of S-type were less active than P-type ones. The highest activity was observed forNaja oxianacobra cardiotoxin 2, which is of P-type and belongs to group II. For the first time, the effects of CaTxs of different groups and types on the cardiomyocytes were studied, and the data obtained showed that the CaTx toxicity to cardiomyocytes depends on the structures both of the N-terminal and central polypeptide loops.