The mutualistic interactions between the oleaginous yeast Lipomyces starkeyi and the green microalga Chloroidium saccharophilum in mixed cultures were investigated to exploit possible synergistic effects. In fact, microalga could act as an oxygen generator for the yeast, while the yeast could provide carbon dioxide to microalga. The behavior of the two microorganisms alone and in mixed culture was studied in two synthetic media (YEG and BBM + G) before moving on to a real model represented by the hydrolysate of Arundo donax, used as low-cost feedstock, and previously subjected to steam explosion and enzymatic hydrolysis. The overall lipid content and lipid productivity obtained in the mixed culture of YEG, BBM + G and for the hydrolysate of Arundo donax were equal to 0.064, 0.064 and 0.081 glipid·gbiomass−1 and 30.14, 35.56 and 37.22 mglipid·L−1·day−1, respectively. The mixed cultures, in all cases, proved to be the most performing compared to the individual ones. In addition, this study provided new input for the integration of Single Cell Oil (SCO) production with agro-industrial feedstock, and the fatty acid distribution mainly consisting of stearic (C18:0) and oleic acid (C18:1) allows promising applications in biofuels, cosmetics, food additives and other products of industrial interest.