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Hydrogen as a clean energy source has a great attention worldwide due to decreasing fossil fuel reserves and rising environmental pollution. Although many types of wastewater have been tested as a cheap substrate in biohydrogen production, the bulgur processing industry wastewater which contains high organic matter has not yet evaluated. This study highlights bulgur processing industry wastewater as a potential substrate for biohydrogen production by dark fermentation. The effects of anaerobic sludge pretreatment methods (thermal and ultrasonic), applying durations (5 and 15 min) for these pretreatment methods, and the ratio of inoculum: substrate (I:S) (1:2, 1:3, 1:4, 1:6 gVS.g⁻¹VS) were investigated in lab-scale batch reactors (at 35°C, pH = 5.5, 16 days of incubation time). Biochemical hydrogen potential (BHP) test results showed that the maximum cumulative bio-hydrogen production of 90.83 mL H₂ g …