Fibrocapsa japonica (Raphidophyceae) is a cosmopolitan species frequently associated with harmful algal blooms (HABs) and fish mortality events, representing a potential threat to aquaculture and coastal ecosystems. This study provides the first comprehensive morphological, phylogenetic, pigmentary, and toxicological characterization of F. japonica strains isolated from Argentina. Light and transmission electron microscopy confirmed key diagnostic features of the species, including anterior flagella and the conspicuous group of mucocyst in the posterior region. Phylogenetic analysis based on the LSU rDNA D1–D2 region revealed monophyletic relationships with strains from geographically distant regions. Pigment analysis by HPLC identified chlorophyll-a (62.3 pg cell⁻¹) and fucoxanthin (38.4 pg cell⁻¹) as the main dominant pigments. Cytotoxicity assays using RTgill-W1 cells exposed for 2 h to culture supernatants and intracellular extracts showed strain-specific effects. The most toxic strain (LPCc049) reduced gill cell viability down to 53% in the supernatant exposure, while LC₅₀ values ranged from 1.6 × 10⁴ to 4.7 × 10⁵ cells mL⁻¹, depending directly on the strain and treatment type. No brevetoxins (PbTx-1, -2, -3, -6, -7, -8, -9, -10, BTX-B1 and BTX-B2) were detected by LC–MS/MS, suggesting that the cytotoxicity may be linked to the production of reactive oxygen species (ROS), polyunsaturated fatty acids (PUFAs), or hemolytic compounds, as previously hypothesized in the literature. These findings offer novel insights into the toxic potential of F. japonica in South America and underscore the need for further research to elucidate the mechanisms underlying its ichthyotoxic effect.