An adequate supply of food sources with high levels (i.e., weight proportion of total fatty acids) and contents (i.e., absolute amount per mass) of long chain polyunsaturated fatty acids such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are important for ecosystems. Therefore, the supply of EPA and DHA from basal food sources is a useful indicator of ecosystem health. To determine whether EPA and DHA levels and contents in fish can be used as indicators of EPA and DHA supply in lakes, five dominant species of fish and basal food sources (seston and sediment) were collected monthly from June to November from 2016 to 2021 from Lake Hachiro, Japan. Seston and Hypomesus nipponensis were collected from 12 lakes (one collection per lake) with varying seston contents in EPA and DHA. The trends of EPA and DHA in all fish species were similar to those of the basal food sources. Correlation analysis showed that the EPA levels were strongly correlated between fish and seston; moreover, the correlation coefficient increased when a 1- or 2-month moving average was applied to the basal food sources, suggesting that fish represent a time-integrated supply of EPA and DHA. EPA levels of H. nipponensis had the highest correlation coefficients with seston among all fish species. EPA levels of H. nipponensis were significantly correlated with those of seston among lakes. The results of this study suggest that H. nipponensis is a useful indicator of EPA and DHA supplies in lake ecosystems.