The Microtox toxicity and Oxygen Uptake Rate (OUR) inhibition tests were conducted to monitor the variation of toxicity during ozonation of 2-chlorophenol (2-CP), 3-chlorophenol (3-CP) and 4-chlorophenol (4-CP) under neutral conditions. The results revealed that the oxidized 2-CP solution exhibited new toxicity to pure bacteria and mixed microorganisms in the early stage of ozonation. The largest inhibition of OUR appeared at one mol of applied ozone dosage per mol of initial 2-CP, and the percentage of inhibition was 63.8%. In addition, ozonated 3-CP and 4-CP also significantly induced new aqueous toxicity, if these toxic intermediates were not further ozonated. Comparing the variation of toxicity and the hydroxylated/chlorinated intermediates formed, 3-chloro-catechol, 2-chloro-2, 4-hexadienedioic acid and the dimmer compounds may be related to the sources of toxicity during the ozonation of 2-CP.