Background: Cell-free mitochondrial DNA (cf-mtDNA) in body fluids has attracted much attention for the purpose of monitoring disease because of the clinical advantages. This study investigated whether the cf-mtDNA content in human follicular fluid samples was associated with oocyte and embryo developmental competence. Methods: We collected 225 individual follicular fluid samples from 92 patients undergoing conventional in vitro fertilization (n = 53) or intracytoplasmic sperm injection (n = 39). cf-mtDNA and cell-free nuclear DNA (cf-nDNA) were measured using real-time quantitative PCR for the ND1 and beta-globin genes. Multivariate logistic regression and linear regression were used to analyze data. Results: The relative cf-mtDNA content (cf-ND1/cf-beta-globin ratio) in follicular fluid was significantly lower in the group showing blastocyst development than in the non-blastocyst group (P = 0.030). Additionally, the relative cfmtDNA content was significantly and positively correlated with the age of the female patient (P = 0.009), while the relative cf-mtDNA content for older women (>= 38 years old) with anti-Mullerian hormone (AMH) = 1.1 ng/ml was significantly higher than in those with AMH > 1.1 ng/ml (P < 0.05). The cf-nDNA content was significantly positively correlated with the antral follicle count (P = 0.012), and significantly negatively correlated with both the number of days of stimulation and the total dose of gonadotropin administration (P = 0.039 and P = 0.015, respectively). Neither cf-mtDNA nor cf-nDNA levels in follicular fluid were associated with oocyte maturation, fertilization, or Day 3 embryo morphological scoring. Conclusions: The relative cf-mtDNA content in human follicular fluid was negatively correlated with blastulation and positively correlated with the patient age, indicating that it is a promising bio-marker to evaluate oocyte developmental competence.