Combining winery wastewater (WW) with sewage sludge (SS) in anaerobic co-digestion (AcoD) presents a promising approach for effective waste management and enhanced energy recovery. This study aimed to evaluate the efficiency and changes in microbial communities during the AcoD of WW with SS at 35 °C, using a mix of batch and semi-continuous testing methods. In the batch experiments, the highest hydrolysis rate (0.19 day−1) and methane production (285.61 ± 4.83 mL/g VS) were achieved at a WW:SS ratio of 3:2, exceeding those of SS mono-digestion by 126 % and 113 %, respectively. Furthermore, the semi-continuous experiments revealed a significant 30.0 % boost in the electron transport system activity and a notable 25.8 % increase in coenzyme F420 activity within the AcoD of WW with SS. Microbial analysis illustrated that AcoD of WW with SS significantly enriched the prevalence of potential genera associated with direct interspecies electron transfer (DIET), such as Longilinea, Bellilinea (genus of Chloroflexi), Syntrophomonas, Pseudomonas, Methanosarcina, and Methanobacterium species. Additionally, semi-continuous experiments demonstrated that AcoD facilitated interspecies hydrogen transfer between syntrophic bacteria and hydrogenotrophic methanogens, resulting in increased methane yield. Co-digestion of WW with SS is a more economical strategy that significantly enhances the stability of microbial communities through the use of complementary feedstocks. These results may offer valuable guidance for the efficient resource utilization of co-digestion processes involving WW and SS.