Lactic acid (LA) production from sugar mixture derived from lignocellulosic rice straw employing co- culture system of thermotolerant and inhibitor tolerant DGB and DGS15 was carried out. In minimal media, both the strains of DGB and DGS15 worked together by efficiently utilising glucose and xylose respectively. Response Surface Methodology (RSM) was used for optimisation of pretreatment of rice straw to achieve maximum yield of 50.852 g/L total reducing sugar (TRS) from 100 gm of rice straw biomass. Pretreatment of rice straw resulted in its delignification, as confirmed by FTIR spectroscopy, since the peak at 1668 cm disappeared due to removal of lignin and scanning electron microscopy (SEM) revealed disruption in structural and morphological features. Crystallinity index ( of treated rice straw increased by 15.54% in comparison to native biomass. DGB and DGS15 individually yielded 0.64 g/g and 0.82 g/g lactic acid respectively, where as their co-cultivation led to effective utilisation of both glucose and xylose within 15 h (70%) and complete utilisation in 48 h, producing 49.75 g/L LA with a yield of 0.98 g/g and productivity of 1.036 g/L/h, and resulting in reduction in fermentation time. Separate hydrolysis of rice straw and co-fermentation (SHCF) of hydrolysates by spp. enhanced the production of lactic acid, can circumvent challenges in biorefining of lignocellulosic biomass.