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Benthic dinoflagellates of the Coolia genus have been associated with cytotoxicity and lethal and sublethal effects on marine species. This study aimed to assess the harmful effects of C. cf. canariensis phylogroup II (PII) and C. malayensis strains through bioassays. Experimental exposures (24, 48, and 72 h) of Artemia salina nauplii to Coolia species (330–54,531 cells mL−1) were performed independently. When a concentration-dependent response was achieved, additional experiments were carried out to evaluate the cell-free medium toxicity. The two Coolia species were harmful to Artemia nauplii, inducing significant mortality and sublethal responses. Coolia cf. canariensis PII was the most toxic species, inducing significant lethality at lower concentrations and shorter exposure times, followed by C. malayensis. Only the survival curves achieved after 24 and 48 h of exposure to C. cf. canariensis PII fitted to a concentration–response curve with valid LC50s of 18,064 and 19,968 cells mL−1, respectively. Moreover, extracellular compounds (i.e., culture filtrates) of C. cf. canariensis PII induced significant mortality to nauplii after 48 and 72 h. The toxicity of C. cf. canariensis PII was demonstrated for the first time using bioassays, and it was surprisingly higher than that of the C. malayensis strain, which was previously demonstrated to induce biological activity at the cellular and subcellular levels. Our findings highlight the harmful and lethal effects induced by Coolia cells and the importance of bioassays for toxicity assessments.