There is evidence that infarcted myocardium contributes to the increase of cell-free DNA levels (cfDNA). We studied the effect of different human DNA fragments on the rate of contraction of the electrically paced neonatal rat ventricular myocytes in culture (spontaneously hypertensive line SHR). AT-rich fragments of the human satellite 3 tandem repeat (1q12 region) at a concentration of 1 ng/mL increase the frequency of cardiomyocyte contractions by 2–2.5 times. GC-rich fragments of the rDNA at 0.5 ng/mL decrease the cardiomyocyte contraction frequency by 1.5–2 times. The serum cfDNA of patients with acute myocardial infarction decreases the frequency of the contraction of cardiomyocytes proportionally to the amount of the rDNA fragments it contains. The rDNA effect is similar to the E. coli DNA effect and is presumably being realized through TLR9. In the presence of the inhibitor of these receptors, rDNA operates the same way as the fragment of the satellite 3—increasing the frequency of the cardiomyocyte contractions. Accumulation of the GC-rich fragments of the cfDNA in the blood of the AMI patients might have an influence on the contractile function of myocardial cells.