Abstract: Xylan the major portion of the hemicellulose of plant cell walls are heterogeneous polysaccharides. Xylanases are enzymes obtained from different species of microoragnisms that degrade the xylosidic linkages of xylans backbone producing xylose with other monoresidues. Five Xylanase-producing strains (St1, St2 and So1, So2, So3 ) were isolated from soil and water at Rajshahi region. The strains were isolated on xylan agar media and screened by βxylanolysis method. Zymogram analysis was confirmed their xylanolytic activity. The isolated xylanase-producing strains were further tested by DNS (Dinitrosalisylic acid) method to confirm their xylanase activity. Five of the isolated strains were identified as Paenibacillus on the basis of catalase, oxidase, gram staining and morphological cultural characteristics as well as by different biochemical test. The strains excrete xylanase enzyme extracellularly in the media. Xylanase enzyme was purified from culture supernatant of Paenibacillus sp. by ammonium sulfate precipitation, gel filtration on Sephadex G-100 followed by ion-exchange chromatography on DEAE. The purified enzyme gave single band on SDS-polyacrylamide gel electrophoresis indicating its homogeneity. The molecular weight of purified xylanase was determined by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. The molecular weight of the purified xylanase was 48 kDa. The optimum temperature and pH of the purified enzyme was 55°C and pH 7.0, respectively. The 30 μg-1 disc-1 of the ethyl acetate extract of the cultural broth of Paenibacillus showed no antibacterial activity against the tested pathogenic bacteria but showed poor antibacterial activity at the concentration of 200 μg-1 disc-1 compared to those of standard kanamycin (30 μg-1 disc-1).