Diatoms play a fundamental ecological role as primary producers in aquatic food webs. Among these, a little number of solitary species of the genus Chaetoceros are of great interest as live food for aquaculture, including major taxa like Chaetoceros calcitrans which have been well studied in terms of growth and essential lipid content. In contrast, the globally distributed species Chaetoceros tenuissimus has not been investigated as a potential live prey for aquaculture. For a preliminary analysis of the effect of culture conditions on growth and biomass content of a new strain of C. tenuissimus (CT16ED, hereafter CTEN) isolated from a Mediterranean lagoon, we first considered the light regime, comparing cultivation under a light–dark (L:D) cycle (12:12 h L:D photoperiod) and under continuous light, which conditions were also applied to a C. calcitrans strain, CCAP1085/3 (CCAL). Both strains had a similar growth rate under continuous light, but growth rate was lower in CTEN than in CCAL under the L:D cycle. Photosynthetic pigments content was higher under the L:D cycle than under continuous light in both strains, and higher pigment content was found in CCAL than in CTEN, indicating different photoacclimation. The lipid content of CTEN did not change markedly with photoperiod or growth phase, unlike CCAL. Both strains had high proportions of essential lipids, particularly n-3 polyunsaturated fatty acids, including EPA and DHA, but these varied less in CTEN than in CCAL. CTEN was richer in DHA (22:6n-3) with a more balanced DHA:EPA ratio. From these results, a potential interest of CTEN for use in aquaculture is discussed, with regard to its nutritional quality (essential lipid content), its growth, and its high-light niche offering possibilities for cultivation in outdoor systems in the Mediterranean area.