Eucheuma cottonii waste seaweed has high cellulose content. Therefore, it could be potentially used as a raw material for biodegradable films to replace plastic. A plastic film is its moisture resistance, and this property allows plastic films to be used as packaging materials and biodegraded by microbes. This research aims to obtain a concentration of glycerol and Carboxy Methyl Cellulose (CMC) to obtain the best biodegradable film characteristics from E. cottonii seaweed waste. This study was conducted in factorial by using a complete randomized block design with two factors: glycerol concentration and CMC concentration. Each treatment has three levels and three replications (3 × 3). The first factor was glycerol concentration: 0.25% (G1), 0.5% (G2), and 0.75% (G3). The second factor included concentrations of 1% CMC (C1), 2% CMC (C2), and 3% CMC (C3). Then, Tensile Strength (TS), thickness, solubility, and elongation were observed. Functional group analysis was conducted by Fourier-transform infrared spectroscopy and biodegradability test. The results showed that the addition of glycerol concentrations of 0.5 and 0.7% and CMC from 1 to 3% produced tensile strengths of 23–39 MPa. These values are proportional to the tensile strength of Poly Tetra Fluoro ethyne (PTFE) and Poly Propylene (PP) synthetic plastics released by Dotmar Engineering Plastics. The biodegradability test showed that the produced biodegradable films decomposed after 14 days.