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Mesta crop biomass is one of the unexplored lignocellulosic feedstocks for second-generation bioethanol production. The compositional analysis of two mesta species, namely Hibiscus sabdariffa and H. cannabinus were carried out which revealed that the mesta contains 41.89–46.50% cellulose that can be saccharified to release fermentative sugar. Among different physical and physico-chemical pretreatment methods employed, cold alkali (2%) treatment showed maximum cellulose enrichment (38.71%) and significant lignin removal (27.27% of total). Optimization of saccharification process for pretreated mesta biomass was carried out using Box–Behnken design. Based on the observations, use of 4.02% substrate loading, 15.02 FPU/mL of cellulase from psychrotolerant Aspergillus niger SH3, and 5.01 IU/gds of β-glucosidase from Pseudomonas lutea BG8, led to 533.25 mg/gds sugar release from the …