A Design of Experiments was adopted to assess the optimal conditions for methane production from the macroalgae Sargassum sp. co-digested with glycerol (Gly) and waste frying oil (WFO). Three variables were tested:% total solids of algae (%TSSargassumsp.), co-substrate concentration (gGly/WFO L-1); and, co-substrate type (Gly or WFO). The Biochemical Methane Potential (BMP) of Sargassum sp. was 181±1 L CH4 kg-1 COD. The co-digestion with Gly and WFO increased the BMP by 56% and 46%, respectively. The methane production rate (k), showed similar behaviour as the BMP, increasing 38% and 19% with Gly and WFO, respectively. The higher BMP (283±18 L CH4 kg-1 COD) and k (65.9±2.1 L CH4 kg-1 COD d-1) was obtained in the assay with 0.5% TS and 3.0 gGly L-1. Co-digestion with glycerol or WFO is a promising process to enhance the BMP from the macroalgae Sargassum sp.