This work aimed to investigate the effect of the initial pH, combination of food to microorganism ratio (F/M) and initial pH, substrate pre-treatment and different inoculum sources on the dark fermentative biohydrogen (H2) yields. Three model complex waste biomasses (food waste, olive mill wastewater (OMWW) and rice straw) were used to assess the effect of the aforementioned parameters. The effect of the initial pH between 4.5 and 7.0 was investigated in batch tests carried out with food waste. The highest H2 yields were shown at initial pH 4.5 (60.6 ± 9.0 mL H2/g VS) and pH 5.0 (50.7 ± 0.8 mL H2/g VS). Furthermore, tests carried out with F/M ratios of 0.5, 1.0 and 1.5 at initial pH 5.0 and 6.5 revealed that a lower F/M ratio (0.5 and 1.0) favored the H2 production at an initial pH 5.0 compared to pH 6.5. Alkaline pre-treatment of raw rice straw using 4% and 8% NaOH at 55 °C for 24 h, increased the H2 yield by 26 and 57-fold, respectively. In the dark fermentation of OMWW, the H2 yield was doubled when heat-shock pre-treated activated sludge was used as inoculum in comparison to anaerobic sludge. Overall, this study shows that the application of different operating parameters to maximize the H2 yields strongly depends on the biodegradability of the substrate.