Hydrogen is an important source of energy and is considered as the future energy carrier post-petroleum era. Nowadays hydrogen production through various methods is being explored and developed to minimize the production costs. Biological hydrogen production has remained an attractive option, highly economical despite low yields. The mixed-culture systems use undefined microbial consortia unlike pure-cultures that use defined microbial species for hydrogen production. This review summarizes mixed-culture system pretreatments such as heat, chemical (acid, alkali), microwave, ultrasound, aeration, and electric current, amongst others, and their combinations to improve the hydrogen yields. The literature representation of pretreatments in mixed-culture systems is as follows: 45–50% heat-treatment, 15–20% chemical, 5–10% microwave, 10–15% combined and 10–15% other treatment. In comparison to pure-culture mixed-culture offers several advantages, such as technical feasibility, minimum inoculum steps, minimum media supplements, ease of operation, and the fact it works on a wide spectrum of low-cost easily available organic wastes for valorization in hydrogen production. In comparison to pure-culture, mixed-culture can eliminate media sterilization (4 h), incubation step (18–36 h), media supplements cost ($4–6 for bioconversion of 1 kg crude glycerol (CG)) and around 10–15 Millijoule (MJ) of energy can be decreased for the single run.