This study investigated the utilization of Pistia stratiotes for biohydrogen production via a dark-fermentation process. The aquatic plant was subjected to acid-hydrolysis using H2SO4: 3.0% (v/v) for 40 min, resulting in sugar yield: 122.2 ± 5.2 mg/g. The optimum culture pH was 5.5, achieving hydrogen yield (HY): 2.46 ± 0.14 mol-H2/mol-glucose (3.51 ± 0.20 mg-H2/g-dry weight) at fermentation time 8 h, temperature 25 °C, and substrate-to-biomass (S/X) ratio 1.0 g-COD/g-VSS. The organic mass balance (92–96%) and electron-equivalent balance (92–98%) indicated the reliability of fermentation data. The dominant species included Planctomycetales, Verrucomicrobiales, Clostridiaceae, and Gammaproteobacteria. The phylogenetic analysis confirmed the abundance of hydrogen-producing bacteria such as Bacillus, Clostridium, and Enterobacter. The hydrogenase gene expression provided the highest activity at pH: 5.5 with a cell number 2.53 × 104 copies/ng-DNA compared to pH: 4.5 (6.95 × 103 copies/ng-DNA) and pH: 8.5 (7.77 × 103 copies/ng-DNA). The total cost of the fermentation system including the amortization cost of investment and operating cost was 0.08 $/kg-dry weight (22.8 $/kg-H2 produced).