Along Chile's extensive coastline, multiple harmful algal bloom (HAB) species have been recorded, including toxic, ichthyotoxic, and high-biomass. However, several relevant taxa remain underreported or insufficiently characterized, particularly athecate, small-sized species and members of the Kareniaceae family. In this study, we investigated the diversity of harmful microalgal species that have been previously overlooked along the Chilean coast and evaluate its toxicological properties. A total of 14 strains were isolated from different locations and characterized using genetic, morphological, pigmentary, cytotoxic, and toxin-based analyses. Here, we report for the first time in Chilean waters the presence of Karlodinium veneficum and Takayama helix (Kareniaceae), as well as Gymnodinium aureolum, Biecheleria cincta (Dinophyceae), and the diatom Thalassiosira pseudonana. In addition, Prorocentrum micans and Lepidodinium chlorophorum were further characterized. K. veneficum exhibited the highest cytotoxicity, even in the absence of identified toxins. In contrast, most strains showed low cytotoxicity and no detectable toxins, however, some of these species have been linked to fish mortality, suggesting the involvement of alternative harmful mechanisms, such as modifications of water rheology. These findings substantially broaden the current baseline of HAB diversity in Chile, underscore the need to integrate these small, athecate and historically overlooked taxa into routine monitoring and early-warning programs, and provide fundamental knowledge for risk assessment during HAB events.