Isobutyrate (i-butyrate) is a versatile platform chemical, whose acid form is used as a precursor of plastic and emulsifier. It can be produced microbially either using genetically engineered organisms or via microbiomes, in the latter case starting from methanol and short-chain carboxylates. This opens the opportunity to produce i-butyrate from non-sterile feedstocks. Little is known on the ecology and process conditions leading to i-butyrate production. In this study, we steered i-butyrate production in a bioreactor fed with methanol and acetate under various conditions, achieving maximum i-butyrate productivity of 5.0 mM day⁻¹, with a concurrent production of n-butyrate of 7.9 mM day⁻¹. The production of i-butyrate was reversibly inhibited by methanogenic inhibitor 2-bromoethanesulfonate. The microbial community data revealed the co-dominance of two major OTUs during co-production of i-butyrate and n-butyrate in two distinctive phases throughout a period of 54 days and 28 days, respectively. The cross-comparison of product profile with microbial community composition suggests that the relative abundance of Clostridium sp. over Eubacterium sp. is correlated with i-butyrate productivity over n-butyrate productivity.