Microalgal biotechnology is always in demand for new fast-growing and oil-rich microalgal strains for different industries. A fusiform diatom strain with robust growth attributes was isolated from Coliumo’s Bay in the Biobio Region, Chile, in 2005. In order to determine the taxonomic identity of the strain, partial sequences of 18S rDNA and ITS region were obtained. Sequence analysis revealed 99% identity with strains of Phaeodactylum tricornutum Bohlin emend. The effect of autotrophic (combination of irradiances, nitrate, and phosphate concentrations) and mixotrophic (addition of sodium acetate under low light) culture conditions was evaluated on growth and lipid accumulation. Hierarchical analysis clustered the autotrophic culture conditions in three main groups of common growth and lipid accumulation responses. Independently on the irradiance, the lowest nitrate and phosphate concentrations induced the highest total lipid accumulation (26–29% lipids per biomass dry weight) and the maximum saturated fatty acid ratio (ca. 42% of total fatty acids), without negative effect on growth; conversely, the highest nitrate and phosphate concentrations stimulated growth and PUFA proportion (20–25% of total fatty acids), but not total lipid accumulation (ca. 15% lipids per biomass dry weight). On the other hand, mixotrophic conditions stimulated growth but not lipid accumulation. Saturated and monounsaturated fatty acid fractions increased as acetate was added while PUFA fraction decreased. Physiological plasticity demonstrated by the Chilean strain of P. tricornutum studied, when faced with different growing conditions, makes it a potential candidate for commercial oil production for different markets, such as biodiesel or feed/food.