In this study, four agro-industrial substrates, chicken litter (CL), food waste (FW), wheat straw (WS) and hay grass (HG) were assessed as feedstock for anaerobic digestion (AD) under semi-continuous conditions at organic loading rates (OLRs) of 2.0-3.0 g TS/L. d and hydraulic retention time (HRT) of 20 days. Six different substrate mixtures were prepared such that the C/N ratio of each was 20 or more. Using principal component analysis 68.1% of data variability was explained. Biogas production from CL, as a single substrate, was 181.3 +/- 9.8 mLN biogas/g VSadded at OLR of 2.0 gTS/L. d. The optimum substrates mixture was CL: FW: WS 60: 20: 20, where 73.0%, 167.2% and 116.9% increase in total biogas production at OLR of 2.0, 2.5, 3.0 gTS/L. d, respectively, compared to that from CL, was obtained. Digestate sequential fractionation revealed carbohydrate degradation is an important factor that can explain the variation in performance and production of biogas for feedstocks of balanced C/N ratio.