Base-hydrolyzed sterols and long-chain alcohols of a marine eustigmatophyte, Nannochloropsis gaditana have been studied. Sterol composition of N. gaditana is similar to that of other member of this genus, with predominance of cholest-5-en-3β-ol and 24-ethylcholest-5-en-3β-ol. The C29:2 and C30:1 unsaturated aliphatic alcohols were predominant. Saturated and monounsaturated C28 to C36n-alkyl diols occurred as a mixture of positional isomers with ω16 predominating among the C28 alkyl diols, ω18 among C30, C32, C34 and C36 alkyl diols and ω17 for the odd chain alkyl diols. C28–C32 hydroxy ketones were identified for the first time in a marine organism, with a distribution parallelling the alkyl diol mixtures and suggesting a biosynthetic relation between both alcohol classes. N. gaditana was fed to Brachionus plicatilis and the distribution of alkyl diol positional isomers remained the same in the algae and the rotifer fecal pellets. However, digestion resulted in an increase in C30 diols relative to C32 diols and in an enrichment of unsaturated long-chain alcohols relative to alkyl diols.