Nine microalgal species from the classes Bacillariophyceae, Cryptophyceae, Prymnesiophyceae and Dinophyceae were isolated from Australian waters, cultured to stationary phase and analyzed for their lipid and fatty acid composition and yield. Five species (Pavlova pinguis, Heterocapsa niei, Proteomonas sulcata, Navicula jeffreyi and Thalassiosira pseudonana) produced high proportions of triacylglycerol (TAG: 22–57% total lipid). An unidentified Navicula-like diatom (CS-786), despite having a low TAG content, had the highest EPA yield (5.8 mg L−1), due to high biomass and a high relative proportion of EPA. Heterocapsa niei had the highest DHA yield (2.9 mg L−1), due to a high cellular lipid and DHA content (171 pg cell−1 and 13.7 pg cell−1, respectively) despite its relatively low biomass. The desirable PUFA composition and yield of both diatom CS-786 and H. niei make them potential candidates for optimization of biomass and PUFA production for use as live-feeds in aquaculture. In addition, H. niei may have potential as a source of DHA for other uses. Low proportions (< 1.2%) of 24:6(n−3) accompanied by trace proportions of 24:5(n−6) were detected in most strains, while 28:8(n−3) was found in dinoflagellates and also in the prymnesiophyte P. pinguis. All non-diatomaceous species contained 26:7(n−3) in minor quantities. This is the first time these unusual C24 and C26 PUFA have been reported in microalgae and the first report of C28 PUFA in a microalga other than dinoflagellates. Possible biosynthetic reasons why these might occur in stationary phase cultures are considered and the likely dietary transfer of these PUFA to higher aquatic life is discussed.