The relationships between extracellular polymeric substances (EPS), soluble microbial product production, metal solubility, and methanogenic activity were investigated. The individual, and joint, toxic effects of nickel and cobalt on methanogenic consortia fed with glucose as model substrate were studied using biomethane potential assays. Cobalt was found to be less toxic to methanogens than nickel at each concentration tested, and the combined effects of Ni and Co on methane production in the bimetal experiment was higher than the sum of the effects of each metal alone. The protein content of EPS, and extracellular soluble protein fractions, decreased with increasing concentrations of total metals. Meanwhile, no significant change in response to metal stress was apparent for carbohydrate content of EPS or extracellular soluble carbohydrate. Decreasing protein content of EPS was accompanied by reduced methanogenic activity and an increase in the soluble metal fraction. The strong associations observed between these variables could be due to the critical role of EPS in protecting microbial cells against nickel and cobalt stress, possibly by capturing metal cations through their functional groups, thus reducing metal availability to the microbial cells in the methanogenic consortia underpinning the anaerobic digestion process. Display omitted •Metal toxicity in AD was associated with metal solubility and EPS content.•Solubility and toxicity of Ni was higher than Co at the same total concentrations.•Ni and Co toxicity resulted in lowered EPS and extracellular soluble protein content.•Soluble and EPS carbohydrate contents were not affected by high metal concentrations.