Haslea ostrearia is a marine pennate diatom, is commercially valued for its water-soluble blue pigment, marennine, which gives oysters their characteristic hue. The lipid and fatty acid profile of H. ostrearia have not been extensively studied, particularly with regard to salinity, which is a key factor in the distribution and metabolism of marine microalgae. At first, we determined the halotolerance range and growth optimum of a Black Sea strain of H. ostrearia, showing its ability to divide in a salinity range of 10–45‰ with an optimum around 22‰. Based on these physiological data, two ecologically relevant salinity levels (30 and 20‰, corresponding to the average values for Mediterranean and Black Seas, respectively) were selected for comparative analysis of lipids and fatty acids composition of a cloned H. ostrearia culture. A decrease in salinity resulted in qualitative and quantitative changes in the fatty acid composition; in particular, at lower salinity (20‰), the qualitative composition of the identified acids was approximately half that at 30‰. At 20‰, there was also a decrease in total lipid content was observed, primarily affecting the sterol fraction. These data suggest that reduced salinity triggers a compensatory mechanism in H. ostrearia aimed at facilitating adaptation and survival in hypoosmotic environment. Based on the obtained results, it is likely that H. ostrearia is a species sensitive to decreased salinity. The findings important for assessing the distribution of this species and diatom culturing.