Increasingly, macroalgae are being recognised as a growth opportunity for functional foods and nutritional security in the future. Dominating traits of interest are metabolites that function as anti-inflammatories and are antiproliferative. However, seaweeds from the northern hemisphere dominate this field of research. Australia has a unique flora of macroalgae, and it is poorly understood which species should be targeted for cultivation towards food and health markets. Here, six Australian marine macroalgae were selected for screening of one anti-inflammatory group; n-3 polyunsaturated fatty acids (PUFA). PUFA profiles were determined using gas chromatography-mass spectrometry and multivariate analysis. Thirty-one fatty acids (FA) were identified across the six macroalgal species with C16:0 the dominant FA in all samples, variations across taxa in the saturated FA C10:0, C14:0, C16:0, C18:0 and C20:0 and variations in monounsaturated FA attributed to C16:1 n-7 and C18:1 n-9. For PUFA profiles, all six species had significantly different n-6/n-3 ratios, while the green seaweed Ulva species possessed the lowest n-6/n-3 ratio of 0.4, along with a 2-fold higher C18:3 n-3 to C18:2 n-6 content. Ulva sp. was the only species that contained docosahexaenoic acid. Extracts of both the Ulva sp. and Hormosira banksii showed selective cytotoxicity towards a human pancreatic cancer cell line, while the nonpolar extracts of all six algae species strongly inhibited production of the inflammatory-mediator nitric oxide.