The energy crisis, depletion of fossil fuels, and global waste issue highlight the need for sustainable and eco-friendly energy processes. In this study, the anaerobic co-digestion of sewage sludge (DS), milk sludge (MS), and food waste (FW) with glycerol (GL) from biodiesel production was investigated. Binary co-digestion of MS-GL, DS-GL, and FW-GL, as well as ternary co-digestion of MS-FW-GL and DS-FW-GL, were examined to determine the optimal combinations for biogas production and process stability. Adding 5% GL (v/v) increased methane yields by 88.78%, 405.82%, and 40.03% for binary mixtures and 55.57% and 298.53% for ternary mixtures, respectively. In addition, the ternary mixtures resulted in the accumulation of volatile fatty acids at 2136.96 mg/L and 1843.62 mg/L, respectively, causing a reduction in methane yield compared to that in binary mixtures. GL added to binary and ternary mixtures delayed optimal methanogenic activities, with higher hydrolysis rates and shorter lag times observed in single substrate-contained and binary mixture reactors. Ternary mixtures showed lower hydrolysis rates and longer lag times, indicating that methanogens required more time to adjust to the increased organic content. Co-digestion procedures using different substrate ratios should consider the risk of organic overloads, which can lead to system instability or failure.