In recent years, the replacement of fossil resources with renewable ones has received great interest, especially as regards the production of new valuable bio-products and bio-fuels, in order to replace the traditional petroleum-based ones. In this context, the exploitation of waste biomasses into added-value bio-chemicals is strongly encouraged. Among these ones, the algae ones are attracting considerable attention, in particular macroalgae which cause eutrophication problems in estuaries and lagoons, due to the drastic reduction of dissolved oxygen during their decomposition. This is true for Orbetello lagoon (Italy), where a large amount of algal biomasses is removed every year through an expensive practice, with consequent environmentally serious disposal problems. In this work, for the first time, the acid-catalyzed conversion of two different macroalgae harvested in Orbetello lagoon, Chaetomorpha linum (Muller) Kutzing and Valonia aegagropila C. Agardh, into levulinic acid was studied and optimized, adopting a one-pot hydrothermal treatment, under microwave heating and in the presence of aqueous diluted mineral acids, H2SO4 and HCl. Levulinic acid is a versatile platform chemical, classified by the United States Department of Energy as one of the top-12 promising bio-based building blocks. The effect of the main reaction parameters to give levulinic acid was investigated and discussed, in particular the type and concentration of the acid catalyst, the temperature and the reaction time. The highest levulinic acid yields of 19 wt% for Chaetomorpha linum and 16 wt% for Valonia aegagropila, calculated respect to the weight of the starting dried biomass, were reached. The achieved results are very promising and confirm the significant potential of these green algae as renewable starting feedstocks for levulinic acid production.