Highway road dust is considered to have a high potential of pollution by trace toxic substances derived from automobiles such as heavy metals and PAHs (polycyclic aromatic hydrocarbons) due to heavy traffic volume of highway, which leads to concern about ecotoxicity in the water environment due to these substances. In this study, ecotoxicity tests on highway road dusts were conducted to show toxicity levels of the highway road dusts. And a couple of new concepts of ecotoxicity evaluation index were also proposed, and ecotoxicity fluctuation of highway road dust was calculated using the index. The followings were done and found. The toxicity level of highway road dust differs depending on the seasons such as winter and non-winter. The significant toxicity was not observed, on average, until the dust ratio is more than 11.20% for winter season and more than 55.11% for non-winter season. In addition, the concepts of C-NOEC (Confidence Interval Based NOEC) and F-NOEC (Cumulative Frequency Based NOEC) were shown as new toxicity evaluation indexes related to NOEC (No Observed Effect Concentration), and their availabilities were examined. Furthermore, the toxicity fluctuation characteristics of highway dusts were shown by calculating the risk ratio using these indexes and content ratios of PAHs.