This study details the feeding ecology of arrow squid, Nototodarus gouldi, collected opportunistically from trawlers in waters south-east off Australia in 2007 and 2008. Combined stomach content and fatty acid (FA) signature analyses provided clear evidence of seasonal dietary shifts in prey composition. Teleost fish remains (mainly otoliths) were found in 67% of stomachs with the two mesopelagic planktivorous fish, Lampanyctodes hectoris and Maurolicus muelleri dominating. Cephalopods and crustaceans were supplementary dietary components, with an increased representation in the diet over winter. Digestive gland lipid content was moderate (16.4 ± 8.4% wet weight) and was rich in triacylglycerol and monounsaturated fatty acids. Multivariate analysis of FA profiles grouped arrow squid with profiles of mesopelagic fish and cephalopods, thus supporting the findings of stomach content analysis. Seasonal differences in total lipid content were likely related to summer upwelling events and local changes in productivity, while intraspecific differences in lipid class and FA composition were related to seasonal differences of prey consumption. FA analyses also demonstrated dietary differences associated with gender, size and female maturation. Such relationships demonstrate that the diet of N. gouldi is closely linked to prey size, abundance and availability and possibly also, to key life-history stages.