This study presents the characterization and application of different pretreatment possibilities to optimize simple carbohydrate production from representative feedstock, followed by ethanol production through optimized saccharification steps using suitable microorganisms. Total soluble carbohydrates and carbohydrate yield were evaluated using dilute acid hydrolysis of biomass and reference materials (starch and cellulose) under different conditions. To explore two-factor interactions, the Plackett-Burman design of experiments was used in the statistical screening of variables and then transformed to a full-factorial design based on the significant variables. According to the experimental findings, dilute acid hydrolysis can yield a considerable amount of soluble carbohydrates. The goodness of fit of the model was evaluated using the coefficient of determination. The optimized carbohydrate yields of the biomass were 0.76 g/g cassava peels, 0.35 g/g sugar beet pulp, and 0.36 g/g green macroalgae. Bioethanol production from optimized hydrolysis conditions using Saccharomyces cerevisiae and Scheffersomyces stipitis DSM 3651, led to a maximum ethanol yield of 0.286 g/g from cassava peels and 0.033 g/g from sugar beet pulp, while using Neurospora intermedia DSM 1265 led to a maximum ethanol yield of 0.076 g/g from green macroalgae. Hence, these biomasses are considered valuable feedstock for bioethanol production with cassava peels having the highest potential.