This study examined the concentration of cell-free mitochondrial DNA (cf-mtDNA) in porcine follicular fluid (FF) and explored whether the cfDNA level in the culture medium could reflect mitochondrial dysfunction in cumulus cell-oocyte complexes (COCs). cfDNA concentration was higher in the fluid of small-sized follicles, compared to that in larger follicles. The length of cfDNA ranged from short (152 bp) to long (1,914 bp) mtDNA in FF, detected by polymerase chain reaction (PCR). cfDNA concentration in FF significantly correlated with the mtDNA copy number in FF but not with the number of one-copy gene (nuclear DNA) in FF. When the COCs were treated with the mitochondrial uncoupler, namely carbonyl cyanide m-chlorophenyl hydrazone (CCCP), for 2 h and incubated for 42 h, subsequent real-time PCR detected significantly higher amount of cf-mtDNA, compared to nuclear cfDNA, in the spent culture medium. The mtDNA number and viability of cumulus cells and oocytes remained unchanged. When the oocytes were denuded from the cumulus cells following CCCP treatment, PCR detected very low levels of cfDNA in the spent culture medium of the denuded oocytes. In contrast, CCCP treatment of granulosa cells significantly increased the amount of cf-mtDNA in the spent culture medium, without any effect on other markers, including survival rate, apoptosis of cumulus cells, and lactate dehydrogenase levels. Thus, cf-mtDNA was present in FF in a wide range of length, and mitochondrial dysfunction in COCs increased the active secretion of cf-mtDNA in the cultural milieu.