A thermo-tolerant diatom species has been isolated from Tunisian hot spring water (40°C). The isolated diatom has been molecularly identified and classified into the genus Halamphora. The growth kinetics, lipid content and distribution of fatty acids were assessed at 20 and 30°C temperature levels and constant irradiance in controlled batch cultures (11 days). Halamphora sp. showed better growth (μ = 0.53 day−1) and a higher lipid yield (25% of the dry weight) at a higher temperature (30°C). Under the two temperatures tested, the highest lipid and fatty acid contents were mainly reached during the stationary growth phase. The fatty acid profile showed a significant content of two essential fatty acids, eicosapentaenoic acid (EPA, 20:5n-3) and arachidonic acid (AA, 20:4n-6), reaching ~15% and ~21% of the total fatty acids, respectively, at 20°C and 30°C. The distribution of the different components of the fatty acids showed that EPA and AA were mainly located in the neutral lipid fraction in the stationary phase.