Systemic inflammation is assumed to be the consequence and the cause of atrial fibrillation (AF); however, the underlying mechanism remains unclear. We aimed to evaluate the level of cell-free DNA (cfDNA) in patients with AF and AF mimicking models, and to illuminate its impact on inflammation. Peripheral blood was obtained from 54 patients with AF and 104 non-AF controls, and cfDNA was extracted. We extracted total cfDNA from conditioned medium after rapid pacing to HL-1 cells. Nuclear and mitochondrial DNA were separately extracted and fragmented to simulate nuclear-cfDNA (n-cfDNA) and mitochondrial-cfDNA (mt-cfDNA). The AF group showed higher cfDNA concentration than the non-AF group (12.6 [9.0–17.1] vs. 8.1 [5.3–10.8] [ng/mL], p < 0.001). The copy numbers of n-cfDNA and mt-cfDNA were higher in AF groups than in non-AF groups; the difference of mt-cfDNA was particularly apparent (p = 0.011 and p < 0.001, respectively). Administration of total cfDNA and mt-cfDNA to macrophages significantly promoted IL-1β and IL-6 expression through TLR9, whereas n-cfDNA did not. Induction of cytokine expression by methylated mt-cfDNA was lower than that by unmethylated mt-cfDNA. Collectively, AF was associated with an increased cfDNA level, especially mt-cfDNA. Sparsely methylated mt-cfDNA released from cardiomyocytes may be involved in sterile systemic inflammation accompanied by AF.