Lipid profiles of three strains (Mexico, Australia, Japan) of Chattonella marina (Subrahmanyan) Hara et Chihara were studied under defined growth (phosphate, light, and growth phase) and harvest (intact and ruptured cells) conditions. Triacylglycerol levels were always <2%, sterols <7%, free fatty acids varied between 2 and 33%, and polar lipids were the most abundant lipid class (>51% of total lipids). The major fatty acids in C. marina were palmitic (16:0), eicosapentaenoic (EPA, 20:5ω3), octadecatetraenoic (18:4ω3), myristic (14:0), and palmitoleic (16:1ω7c) acids. Higher levels of EPA were found in ruptured cells (21.4–29.4%) compared to intact cells (8.5–25.3%). In general, Japanese N-118 C. marina was the highest producer of EPA (14.3–29.4%), and Mexican CMCV-1 the lowest producer (7.9–27.1%). Algal cultures, free fatty acids from C. marina, and the two aldehydes 2E,4E-decadienal and 2E,4E-heptadienal (suspected fatty acid-derived products) were tested against the rainbow trout fish gill cell line RTgill-W1. The configuration of fatty acids plays an important role in ichthyotoxicity. Free fatty acid fractions, obtained by base saponification of total lipids from C. marina showed a potent toxicity toward gill cells (median lethal concentration, LC50 (at 1 h) of 0.44 μg · mL−1 in light conditions, with a complete loss of viability at >3.2 μg · mL−1). Live cultures of Mexican C. marina were less toxic than Japanese and Australian strains. This difference could be related to differing EPA content, superoxide anion production, and cell fragility. The aldehydes 2E,4E-decadienal and 2E,4E-heptadienal also showed high impact on gill cell viability, with LC50 (at 1 h) of 0.34 and 0.36 μg · mL−1, respectively. Superoxide anion production was highest in Australian strain CMPL01, followed by Japanese N-118 and Mexican CMCV-1 strains. Ruptured cells showed higher production of superoxide anion compared to intact cells (e.g., 19 vs. 9.5 pmol · cell−1 · hr−1 for CMPL01, respectively). Our results indicate that C. marina is more ichthyotoxic after cell disruption and when switching from dark to light conditions, possibly associated with a higher production of superoxide anion and EPA, which may be quickly oxidized to produce more toxic derivates, such as aldehydes.