Buckminsterfullerene (C(60)) has received great research interest due to its extraordinary properties and increasing applications in manufacturing industry and biomedical technology. We recently reported C(60) could enter bacterial cells and bind to DNA molecules. This study was to further determine how the DNA-C(60) binding affected the thermal stability and enzymatic digestion of DNA molecules, and DNA mutations. Nano-C(60) aggregates and water-soluble fullerenols were synthesized and their impact on DNA biochemical and microbial activity was investigated. Our results revealed that water-soluble fullerenols could bind to lambda DNA and improve DNA stability remarkably against thermal degradation at 70-85 °C in a dose-dependent manner. DNase I and HindIII restriction endonuclease activities were inhibited after interacting with fullerenols at a high dose. Experimental results also showed the different influence of fullerenol and nano-C(60) on their antibacterial mechanisms, where fullerenols contributed considerable impact on cell damage and mutation rate. This preliminary study indicated that the application of fullerenols results in significant changes in the physical structures and biochemical functions of DNA molecules.