We have previously shown that DNA damage induced by doxorubicin was associated with an increase in the level of DNA methylation, 5meC, in mouse embryonic fibroblasts using a developed immunostaining methodology. Doxorubicin (also known as adriamycin), which induces double-strand DNA breaks, has been widely used in cancer therapy. Cytosine modifications are dynamically regulated in the genome during lifespan, but abnormal changes in the 5meC pattern can be associated with diseases in particular cancer progressions and DNA damage. One of the side effects of cancer therapy is that chemotherapeutic agents can damage healthy cells as well as cancerous cells. In this study, we aimed to investigate (i) the effect of doxorubicin at a dose that does not induce DNA damage on cytosine modifications 5meC, 5hmC, and 5fC, and (ii) the effect of a treatment known to be nongenotoxic (room light) on 5meC, 5hmC, and 5fC. We found that 5fC is the most sensitive modification to each treatment experienced in healthy fibroblasts as its level decreased after each condition, and its decrease did not correlate to DNA damage. The results suggest preliminary information on how healthy noncancerous cells can response to chemotherapeutics in terms of cytosine modifications.