The Australian lungfish, Neoceratodus forsteri Krefft, 1870, is a threatened species whose long-term persistence is at risk due to land-use intensification, water resource development, and other human pressures. Changes to the hydrology of rivers has the potential to alter the availability of certain high-quality food resources for this species, that may impact recruitment success, and contribute to population declines. This study analysed the fatty acid (FA) composition of lungfish eggs and fin tissues from two locations upstream and downstream of a large dam in the Brisbane River. We tested the hypothesis that river impoundment and flow alteration associated with the dam have altered the dietary composition and the FA composition of important dietary items for N. forsteri which translates to the body tissues and eggs. The contribution of each food source was estimated with mixing models using carbon and nitrogen stable isotopes. The total FA content in lungfish fin and eggs was significantly higher in downstream sites compared to upstream. Few significant differences in FA contents of the nine potential lungfish food sources were found between sites upstream and downstream of the dam. Stable isotope analyses of lungfish fin tissues revealed the most likely food sources were gastropods, bivalves, and crustaceans, however their relative importance differed upstream and downstream of the dam. Collectively, these results indicate that the dam did not negatively affect food quality for lungfish downstream. The most likely mechanism for potential FA deficiency and subsequent impacts on recruitment success in N. forsteri would be due to changes to the availability of high-quality food sources. This study highlights the need for future research to determine whether the low FA contents we observed in lungfish, is a function of broader environmental changes, or if FA contents are naturally low for this sub-tropical species.