Biphasic acid hydrolysates and enzymatic hydrolysates from carbohydrate-rich Prosopis juliflora, an invasive perennial deciduous shrub of semi-arid regions, were used for bioethanol production. Saccharomyces cerevisiae and Pichia stipitis were used for fermentation of hexoses and pentoses. P. juliflora acid hydrolysate with an initial sugar concentration of 18.70 +/- 0.16 g/L was concentrated to 33.59 +/- 0.52 g/L by vacuum distillation. The concentrated hydrolysate was pretreated and fermented by mono- and co-culture methods either singly or in combination with enzyme hydrolysate and ethanol yields were compared. Monoculture with S. cerevisiae (VS3) and S. cerevisiae (NCIM3455) yielded maximum ethanol of 36.6 +/- 1.83 g/L and 37.1 +/- 1.86 g/L with a fermentation efficiency of 83.94 +/- 4.20\% and 84.20 +/- 4.21\%, respectively, after 36 h of fermentation. The ethanol yield obtained was 0.428 +/- 0.02 g/g substrate and 0.429 +/- 0.02 g/g substrate with a productivity of 1.017 +/- 0.051 g/L/hand 1.031 +/- 0.052 g/L/h, respectively. P. stipitis (NCIM3498) yielded maximum ethanol of 24 g/L with ethanol yield of 0.455 +/- 0.02 g/g substrate and a productivity of 1.004 +/- 0.050 g/L/h after 24 h of fermentation. With concentrated acid hydrolysate as substrate, S. cerevisiae (VS3) produced ethanol of 8.52 +/- 0.43 g/L, whereas S. cerevisiae (NCIM3455) produced 5.96 +/- 0.30 g/L of ethanol. P.stipitis (NCIM3498) produced 4.52 +/- 0.23 g/L of ethanol by utilizing 14.66 +/- 0.87 g/L of sugars. Co-culture with S. cerevisiae (VS3) addition after 18 h of addition of P. stipitis (NCIM3498) to the mixture of concentrated acid hydrolysate and enzyme hydrolysate produced 13.86 +/- 0.47 g/L of ethanol with fermentation efficiency, ethanol yield and productivity of 87.54 +/- 0.54\%, 0.446 +/- 2.36 g/g substrate and 0.385 +/- 0.014 g/L/h, respectively. Hence, it is concluded that co-culture with S. cerevisiae and P. stipitis is feasible, further scaling up of fermentation of P. juliflora substrate for bioethanol production.