Parthenium hysterophorus world's seven most devastating and hazardous weeds, and is abundantly available in several parts of the world. This study treats the subject of effective utilization of this waste biomass (which has cellulose content of 45.2 +/- 1.81% w/w) for biofuels production. We have presented a comprehensive and comparative assessment of numerous pretreatment strategies for P. hysterophorus, comprising of all major physical, chemical and physicochemical methods. The yardstick of assessment has been amount of fermentable sugars released during the pretreatment and the post-treatment enzymatic hydrolysis of pretreated biomass. Carboxymethylcellulase (1.0 U/mg, 1.7 mg/mL) produced by an isolate Bacillus amyloliquefaciens SS35 and beta-glucosidase (Novozyme 188), have been used for enzymatic hydrolysis of pretreated biomass. Among the different methods employed for pretreatment, the most efficient treatment has been revealed to be autoclaving of biomass at 121 degrees C and 15 psi pressure for 30 min in acidic (1% v/v, H2SO4) environment. Total reducing sugar (TRS) yield during this pretreatment, mainly due to hydrolysis of hemicellulosic fraction of biomass, has been 285.3 mg/g of raw biomass. Further enzymatic hydrolysis resulted in reducing sugar yield of 187.4 mg/g of pretreated biomass (9.37 g/L). The total fermentable sugar (TFS) yield from the optimized pretreatment was 397.7 mg/g raw biomass (39.77 g/100 g raw biomass). The effects of different pretreatment methods on biomass structure and complexity were investigated by field emission scanning electron microscopy (FESEM), Fourier transform infrared (FTIR) spectroscopy and X-ray diffraction (XRD) techniques. Comparative assessment of the results with pretreatment and hydrolysis of other conventional agro- and forest residues shows that P. hysterophorus has same potential for being the feedstock for biofuels. These results conclusively demonstrate the utility of P. hysterophorus for sustainable biofuels production. (C) 2014 Elsevier Ltd. All rights reserved.