The growth of microalgae in hypersaline conditions requires that cells accumulate osmoprotectants. In many instances, these are polyols. We isolated the diatom Nitzschia ovalis H. J. Arn. from the saline and alkaline water body Mono Lake (CA, USA). This isolate can grow in salinities ranging from 5 to 120 parts per thousand (ppt) of salt but normally at 90 ppt salinity. In this report, we identified the major polyol osmoprotectant as 1,4/2,5 cyclohexanetetrol by electron ionization-mass spectrometry (EI–MS), 1H, 13C nuclear magnetic resonance spectroscopy (NMR), and infrared (IR) and showed an increase in cellular concentration in response to rising salinity. This increase in the cyclitol concentration was evaluated by gas chromatography of the derived tetraacetylated cyclohexanetetrol obtaining an average of 0.7 fmol · cell−1 at 5 ppt and rising to 22.5 fmol · cell−1 at 120 ppt. The 1,4/2,5 cyclohexanetetrol was also detected in the red alga Porphyridium purpureum. Analysis of the free amino acid content in N. ovalis cultures exposed to changes in salinity showed that proline and lysine also accumulate with increased salinity, but the cellular concentration of these amino acids is about 10-fold lower than the concentration of 1,4/2,5 cyclohexanetetrol. The comparison of amino acid concentration per cell with cyclitol suggests that this polyol is important in compensating the cellular osmotic pressure due to increased salinity, but other physiological functions could also be considered.