Display omitted •Effect of various inoculum pretreatment on microbial community dynamics evaluated.•H2 yield was 70% higher for acid and alkali-treated inoculum than untreated.•The combined impact of heat and CO2 enriched > 90% spore-forming fermentative bacteria.•Clostridial-type fermentation dominated mesophilic H2 production from food waste. This study systematically evaluated and compared different inoculum pretreatment methods to quickly select dark fermentative bacteria from anaerobic sludge for the bioconversion of food waste. The hydrogen (H2) production rate was found to be highest for 'heat + CO2′ treated inoculum at 140.75 ± 2.61 mL/L/h compared to control experiments (60.27 ± 2.61 mL/L/h). At the same time, H2 yield was found to be highest for alkali-treated inoculum at 157.25 ± 7.62 mL/g of volatile solids (VS) added compared to control experiments (91.61 ± 1.93 mL/g VS). Analysis of organic acids suggests a Clostridial-type fermentation with acetate (0.52 to 1.60 g/L) and butyrate (1.69 to 2.42 g/L) being the major by-products. The microbial data analysis showed that Firmicutes (63.64–90.39%), Bacteroidota (1.16–21.88%), and Proteobacteria (2.09–9.93%) were dominant at the phylum level, whereas genus-level classification showed Clostridium sensu stricto 1 (6.37–42.63%), Streptococcus (1.87–28.96%), Prevotella (0.57–16.59%), and Enterococcus (0.56–14.51%) dominated under different experimental conditions.