Chlorine has effectively reduced the prevalence of waterborne diseases, however there are secondary consequences to this public health advancement. Disinfection byproducts (DBPs) are chemicals formed when chlorine reacts with natural organic matter (NOM) in water. A new class of DBPs, halobenzoquinones (HBQs), has recently been identified and data suggests it could be potentially carcinogenic and up to 1000 times more toxic than some regulated DBPs. So far, in vitro studies have assessed HBQ toxicity without taking into account its transformation in cell media into potentially less toxic compounds. This study evaluated the toxic effects of one HBQ, 2,6-DCBQ, and its transformed derivatives on colon epithelial and liver hepatoma cell lines by measuring intracellular reactive oxygen species production and cell viability post-DCBQ exposure. In addition, to better quantify the trade-off between exposure to waterborne pathogens and 2,6-DCBQ, the inactivation of a virus indicator (MS2), and formation of DCBQ were determined in chlorinated surface waters. Dose-dependent toxic effects were observed in both cell lines and transformed DCBQs were observed to be less toxic than their parent compound. MS2 inactivation occurred immediately post-chlorination, but DCBQ was detected simultaneously. Such findings indicate that this compound is toxic to human cells, including colon epithelial cells, which may be pertinent due to the possible association between chlorinated waters and colon cancer. Findings also suggest this DBP may be relevant in developing countries because HBQs may form in point-of-use chlorinated drinking waters. Furthermore, observed reduction in toxicity of media-transformed DCBQs calls current literature on HBQ toxicity into question.