Two single cell heterotrophs were produced as alternative sources of docosahexaenoic acid (DHA; 22:6 n-3) to fisheries-derived oils. Schizochytrium G13/2S or Crypthecodinium cohnii biomasses, either homogenised (SH, CH, respectively) or non-homogenised (S, C), were tested in gilthead seabream (Sparus aurata) microdiets. In Trial 1 the biomases of both species algae were used in replacement of fish oil, whereas in Trial 2 all dietary lipids, including those derived from squid meal, were replaced by the algal biomass in all diets except for control diet. Larval dietary acceptance, overall survival, air-exposure stress survival, growth and disease resistance were not significantly affected by the source of DHA employed when only fish oil was substituted. However, complete substitution of all dietary lipids by the algal biomass, resulted in reduced growth and survival which was apparently related to other dietary imbalances besides simply DHA content, such as changes in the DHA:EPA (eicosapentaenoic acid; 20:5 n-3) ratio. After 15 and 21 d feeding the experimental diets to S. aurata larvae in Trial 1 (0.1 mg initial larval dry weight) and Trial 2 (0.15 mg initial larval dry weight), respectively, the algal biomass inclusion or homogenisation did not significantly affect the DHA deposition in fish body (14.4 ± 0.3% total fatty acids ± SEM and 28.4 ± 0.4, average values for Trials 1 and 2, respectively). Higher arachidonic acid levels were found in larvae fed Schizochytrium sp. diets in Trial 2 and were related to the retro-conversion from docosapentaenoic acid (DPA; 22:5 n-6) which was high in these diets. These results show the potential of single cell heterotrophs as alternative DHA sources for fish oil in microdiets for gilthead seabream but also point out the necessity of EPA sources to completely replace fisheries-derived oils.