A brown alga Saccharina japonica and rice straw are attractive feedstock for microbial butyric acid production. However, inefficient fermentation of mannitol (a dominant component in S. japonica) and toxicity of inhibitors in lignocellulosic hydrolysate are limitations. This study demonstrated that mixed biomass with S. japonica and rice straw was effective in butyric acid production over those restrictions. Mannitol was consumed only when acetic acid was present. Notably, acetic acid was not produced but consumed along with mannitol. By mixing S. japonica and rice straw to take advantage of glucose and acetic acid in rice straw, Clostridium tyrobutyricum effectively consumed mannitol by utilizing acetic acid in hydrolysate and acetic acid derived from glucose with the enhanced butyric acid production. Furthermore, cell growth was restored owing to the decreased inhibitor concentration. The results demonstrate the potential of butyric acid production from mixed biomass of macroalgae/lignocellulose overcoming the drawbacks of single biomass.