Summary Cell-free DNA (cf-DNA) is defined as DNA fragments that are released into the body fluids from apoptosis or necrosis cells, including follicular fluid (FF), which can affect the microenvironment of the oocyte associated with infertility. We aimed to investigate a relationship between apoptosis of cumulus cells (CCs) and cf-DNA levels in FF and clinical outcomes of women undergoing intracytoplasmic sperm injection (ICSI). Therefore, 82 FF samples were collected, and the corresponding CCs were isolated for ICSI procedures. FF cf-DNA concentration was quantified using ALU-quantitative polymerase chain reaction (PCR), and CCs DNA fragmentation index (DFI) was evaluated by the terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labelling (TUNEL) method. We found that cf-DNA and DFI levels were significantly higher in FF and CCs samples related to the age of women ≥37 years compared with the age of women < 37 years. Moreover, in older and younger women, FF cf-DNA and CCs DFI levels were significantly lower when the anti-Müllerian hormone (AMH) level was > 1.1 ng/ml compared with when AMH ≤ 1.1 ng/ml. In addition, patients with a low number of retrieved oocytes ≤ 6 had significantly higher levels of CCs DFI and FF cf-DNA than women with a higher number of retrieved oocytes > 6. Additionally, we observed that higher levels of cf-DNA and DFI were associated with poor oocyte maturity and poor embryo quality. Finally, cf-DNA and DFI levels were significantly lower in pregnant women than in non-pregnant ones. We conclude that DFI and cf-DNA levels in the oocyte microenvironment could have potential use in evaluating oocyte and embryo developmental competence.