The economically viable large-scale production of the pure isoforms of the surfactin biosurfactants, involving bacterial – Bacillus subtilis – fermentation of biomass hydrolysate feedstock, relies on the types of bacterial strains, optimization of the fermentation processing parameters, differences in the composition of the carbon and nitrogen in the bacterial media, and the chromatography techniques used for isolation of the isoforms. Here, we biosynthesized the surfactin isoforms in their mixture forms through fermentation of biomass hydrolysates at 2 wt.% carbohydrate content. The surfactin isoforms were assessed for their surface-active properties and toxicity. The enzyme hydrolysates considered were from switchgrass, soyhull (fiber), alfalfa, and bagasse. The isoform mixtures obtained after fermentation of the hydrolysates and, glucose as a control, were concentrated using chromatography columns, and characterized for molecular weights (MWs) and relative distribution using LCMS. The isoform mixtures, obtained in different fermenters (5- and 15-L) and, for different hydrolysates, invariably constituted 5 isoforms with MWs as 992.6, 1006.6, 1020.6, 1034.6, 1048.6, 1062.6 m/z amu, with their relative proportions as 6, 24, 35, 24, and 10 weight % respectively. The surface tension values of all these isoforms, in the absence of electrolytes and at 12 ppt salinity, were similar: 37 (pH 6.5) and 31 (pH 9.5) mN/m. Furthermore, the emulsification index values for the isoforms were also similar: Dispersant-to-Oil ratio as 1:20. The LC 50 for Gulf killifish, Fundulus grandis for these surfactin isoforms ranged between 10 and 20 mg/L; a microbially-produced surfactin variant FA-Glu (Fatty acid Glutamate) was least toxic with LC 50 at ∼100 mg/L. Thus, the surfactin synthesis approach adopted here suggested that pure (>95 wt.%) non-toxic isoforms of surfactin biosurfactants can be produced in the forms of their mixtures with surface-active properties similar to those of the pure forms of the surfactin isoforms.