Seaweed has emerged as an alternative feedstock for the production of a number of renewable fuels. However, the commercialization of seaweed to biofuels requires optimization to improve yields, before scale-up is feasible. The resistance of the algal cell wall to hydrolysis is a limiting factor in fermentation, thus an optimal pretreatment process would enhance the bio-digestibility of the algal biomass, increase accessibility of biomass to hydrolytic enzymes and, ultimately, improve biofuel yields. This study focused on measuring the amount of macromolecules, reducing sugars (RS), total lipids (TL), and total proteins (TP) released from two native seaweed species in Ireland (Laminaria digitata and Saccharina latissima) in response to a range of chemical, mechanical, and thermal pretreatments. The overall results showed that all pretreatment methods enhanced the release of macromolecules to different extents when compared to untreated controls. Among the set of pretreatments studied, a combination of milling and 4% nitric acid at 130 degrees C for 2hrs had the greatest effect in releasing RS and TL from both seaweed species. Findings from this study highlight the importance of seaweed pretreatment and its potential use in the optimization of biofuel production.