Fish-killing algal species are responsible for important global economic losses to the finfish industry. Chile is the world’s second-largest salmon exporter, and fish-killing algal blooms have widely impacted its production in the last decades. The lack of standardized analytical methods to quantify and characterize the so-called “ichthyotoxins” has hindered our understanding of the underlying ichthyotoxic modes of action. The novel application of a highly sensitive and reproducible fish RTgill-W1 cell line-based assay has allowed significant progress in the field. In this study, the ichthyotoxic potency of the main microalgae species, which has been reported in fish-killing events in the historical Chilean monitoring programs, was assessed. The dinoflagellate Karenia selliformis was the most ichthyotoxic species against the RTgill-W1 (cell viability down to 8%), representing the major threat for the local salmon industry. In comparison, the raphidophyte Heterosigma akashiwo and the dinoflagellate Prorocentrum micans were the least toxic (gill cell viability down to 81 and 89%, respectively). Importantly, ichthyotoxic flagellates were more toxic to fish gill cells upon rupture. These results have important implications for the mitigation and management of algal blooms by the salmon industry.