In this report, Polyurethane (PU) degrading microorganisms (fungi and bacteria) were isolated from soil through enrichment. The isolated fungal strain was identified by examination of colony morphology i.e. color, size and colony diameter and shape, color, size and structure of conidia, hyphae, conidiophores and conidial head as Aspergillus tubingensis. PU films incubated for one month on MSM-Agar plates inoculated with A. tubingensis demonstrated visible signs of degradation in terms of changes in color and flexibility. Thick mycelial growth and adherence of fungal biomass with surface of PU was confirmed by scanning electron microscopy (SEM). Fourier transformed infra-red spectroscopy (FTIR) spectrum of the treated PU film, when compared to that of untreated control revealed changes in important functionalities. Two bacterial strains isolated from the same soil were identified as Bacillus subtilis MZA-75 and Pseudomonas aeruginosa MZA-85 by colony morphology, microscopy, biochemical characterization and 16S rRNA gene sequence analysis. The degradation of PU film pieces exposed to both strain MZA-75 and MZA-85 was investigated by SEM, FTIR and gel permeation chromatography (GPC). SEM micrographs of PU film pieces, treated with strains MZA- 75 and MZA-85, showed alterations in the morphological features of surface. FTIR spectrum demonstrated rise in organic acid functional groups and fall in ester functionality. GPC results revealed increase in polydispersity, which shows that long chains of polyurethane polymer are cleaved into shorter chains by microbial action. Increase in cell growth and CO 2 concentration detected through Sturm Test, in comparison to control further elaborate the degradative capability of strains MZA-75 and MZA-85. MZA-85 was found capable of producing cell associated esterase measured on the basis of p-Nitrophenyl acetate (pNPA) hydrolysis assay. Time course study for cell associated esterase in the presence and absence of PU in MSM broth revealed that this enzyme is induced by the presence of PU in the medium. Crystal violet staining and SEM results shows that MZA-85 forms biofilm on the surface of PU. In case of MZA-75 increase in both cell bound and extracellular esterases was observed in the presence of PUR films in MSM as compared to control when analyzed through p-Nitrophenyl acetate (pNPA) hydrolysis assay. PUesterase was purified from xiithe MZA-75 by using Sephadex G-75 column chromatography. The purified enzyme gave single band on SDS-PAGE corresponding to molecular weight 51 KDa. Substrate specificity analysis was done using p-Nitrophenyl acyl esters of varying carbon numbers. Maximum esterolytic activity was observed in case of p-Nitrophenyl butyrate (C 4 ). Analysis of the cell free supernatant by GC-MS, revealed that 1, 4-butanediol and adipic acid monomers were produced as result of degradation of PU by both MZA-75 and MZA-85 and both the strains were capable of utilizing these intermediates as carbon source. Both MZA-75 and MZA-85 are subject to further studies to understand their interaction with PU completely, which may be helpful in PU bioremediation and biochemical monomer recycling from PU wastes.