The inhibition of acetate under acidic pH is an ideal way to reduce methanogenesis in mesophilic mixed culture fermentation (MCF). However, the effects of acetate concentration and acidic pH on methanogenesis remain unclear. Besides, although hydrogenotrophic methanogens can be suitable targets in MCF, they are generally ignored. Therefore, we intentionally enriched hydrogenotrophic methanogens and found that free acetic acid (FAA, x) concentration and specific methanogenic activity (SMA, y) were correlated according to the equation: y = 0.86 x 0.31/(0.31 + x) (R-2 = 0.909). The SMA was decreased by 50% and 90% at the FAA concentrations of 0.31 and 2.36 g/L, respectively. The coenzyme M concentration and relative electron transport activity agreed well with the FAA concentration. Moreover, the methanogenic activity could not be recovered when the FAA concentration exceeded 0.81 g/L. These findings indicated that neither acetate nor acidic pH, but FAA was the key factor to inhibit methanogenesis in MCF.