This study investigated the effect of acetate and methanol as co-substrates on anaerobic biodegradation of total petroleum hydrocarbons (TPHs, C-10-C-40) in marine sediment. The findings evidenced that the degradation of TPH can be enhanced by adding acetate or methanol. The addition of acetate was generally more favorable than the addition of methanol for the TPH degradation. Both sulfate reduction and methanogenesis occurred in the acetate-treated sediment. However, the depletion of SO42- inhibited sulfate reduction over the incubation period. Only methanogenesis was prevalent in the methanol-treated sediment within the whole incubation period. The degradation of TPH fractions with higher carbon number ranges (C-31-C-40) was speculated to be more favored under sulfate-reducing condition, while TPH fractions with lower carbon number ranges (C-10-C-20) were preferentially degraded under methanogenic condition. The 16S rRNA clone library-based analysis revealed that the addition of different co-substrates led to distinct structures of the microbial community. Clones related to sulfate-reducing Desulfobacterales were the most abundant in the sediment dosed with acetate. Clones related to Clostridiales predominated in the sediment dosed with methanol. Acetoclastic methanogens were found to be the predominant archaeal species in the sediment dosed with acetate, while both acetoclastic methanogens and hydrogenotrophic methanogens accounted for large proportions in the sediment dosed with methanol. The results obtained in this study will contribute to more comprehensive knowledge on the role of acetate and methanol as co-substrates in biostimulation of petroleum-hydrocarbon-contaminated marine sediment.