The potential effects of the fullerene C60 nanoparticle (C60) as well as virgin olive oil (VOO) against the cyclophosphamide- (CP-) induced cytotoxic and mutagenic effects were evaluated by two main methods: molecular intersimple sequence repeat (ISSR) assay and cytogenetic biomarkers. Thirty adult male rats were divided to five groups (control, CP, C60, CP + C60, and CP + VOO). CP was i.p. injected with a single dose of 200 mg/kg; C60 and VOO were given orally (4 mg/kg dissolved in VOO and 1 ml, resp.) in alternative days for 20 days. The ISSR analysis revealed an increased in the DNA fragmentation level for liver and heart tissues represented by 21.2% and 32.6%, respectively, in the CP group. The DNA polymorphism levels were modulated and improved in CP + C60 (8.9% and 12%) and CP + VOO (9.8% and 12.7%) for hepatic and cardiac tissues, respectively. The bone marrow cytogenetic analysis revealed that C60 and VOO had significantly decreased the frequency of CP-induced chromosomal aberrations (chromosomal ring, deletion, dicentric chromosome, fragmentation, and polyploidy). Fullerene C60 and VOO have ability to reduce DNA damage and decrease chromosomal aberrations. In conclusion, fullerene C60 and VOO have protective effects against the CP-induced mutagenicity and genotoxicity. Fullerene C60 and VOO open an interesting field concerning their potential antigenotoxic agents against deleterious side effects of chemotherapeutics.