- Abstract Anaerobic digestion (AD) of sulfate-rich brackish aquaculture sludge (BAS) is an effective method to reduce negative environmental impact and produce biogas. However, the mechanisms of electron-acceptor utilization during AD of BAS, and the effects of elevated sulfate, nitrate and nitrite levels on biogas production, have never been investigated. The current study is the first to demonstrate the mechanisms of electron-acceptor utilization in BAS under methanogenic conditions. Nitrate, nitrite and iron were almost completely depleted during the first day of AD, and methane production began only when these electron acceptors were removed. In contrast, sulfate reduction also started immediately, but took 15–28 d, dependent on the treatment, and largely coincided with methane production. The volume of produced biogas, the lag phase for methane production and methane content in the biogas were affected by the inhibition of sulfate reduction and by the increase in nitrate and nitrite concentrations. However, the average methane-production rates in all treatments were similar. Our data suggest that methanogenesis in BAS under naturally high sulfate levels results in the highest possible methane-producing capability of the system; no additional manipulations are required to further increase methane production, suggesting the robustness of AD of BAS under the currently reported conditions.