Seven algal species were used to conduct toxicity assays with 12 herbicides to determine differences in species sensitivity. A fluorescence microplate toxicity assay was used as an efficient and economical high-throughput assay. The obtained toxicity data were standardized based on the species sensitivity distribution concept. The most-sensitive individual species differed among herbicides: Desmodesmus subspicatus was most sensitive to chloronitrofen and pendimethalin; Achnanthidium minutissimum was most sensitive to chlorpropham; Nitzschia palea was most sensitive to diquat, glyphosate, and dichlobenil; Navicula pelliculosa was most sensitive to trifluralin; and Pseudanabaena foetida was most sensitive to glufosinate, asulam, and 2,4-D. Surprisingly, Raphidocelis (formerly Pseudokirchneriella) subcapitata, a standard green alga, was not the most sensitive to any of the herbicides. The results clearly showed that a single algal species cannot represent the algal assemblage in terms of sensitivity. Therefore, multispecies algal toxicity data sets are essential for assessing the ecological effect of herbicides.