As concerns regarding water pollution grow, the need increases for a fast and accurate assessment of ecological risk. In this context, many studies have been conducted to identify biomarkers which can sensitively indicate exposure to and effects of various contaminants in a water environment. However, the utility of most such biomarkers in the real water environment is not yet validated. In this paper, we conducted a thorough review of publications that were related to developing or evaluating molecular and biochemical biomarkers of freshwater fish in ecological risk assessment, and evaluated whether these biomarkers of interest could link to the effects on higher biological levels, such as histopathology and above. Biomarkers of interest included those associated with metabolism, oxidative stress, reproduction and endocrine disruption, genotoxicity, and defense against heavy metal exposure. We found that, when used alone, most molecular and biochemical biomarkers are not sufficient to understand the effects of toxic substances in higher biological levels, due to defense or acclimation mechanisms of organisms. Moreover, some biomarkers respond not only to hazardous substances but also to the changes in water quality and disease outbreak. Molecular and biochemical biomarkers may be most useful in understanding the potential biological effects of toxic compounds when used in parallel with relevant endpoints of higher biological levels.