Nanofibers were isolated from bagasse pulp pretreated with dilute hydrochloric acid, dilute sodium hydroxide, cellulase, or xylanase enzymes using high-shear ultrafine grinding and high-pressure homogenization. The effect of the different pretreatments on chemical composition and structure of isolated nanofibers was studied using chemical analyses, X-ray diffraction, and Fourier transform infrared. The dimensions and properties of the isolated nanofibers were followed at the different processing stages using optical microscopy, transmission electron microscopy, atomic force microscopy, and tensile properties (wet and dry). The diameter of the microfibrils was in the range of 7-30 nm for untreated and pretreated bagasse pulps while larger microfibrillar bands (to 150 nm wide) were observed for untreated bagasse pulp than the pretreated pulps (to 90 nm wide). Nanopaper sheets made from nanofibers isolated from alkali- and xylanase-treated pulps showed better wet and dry tensile strength than those made from the other pulps. ; Nanofibers were isolated from bagasse pulp pretreated with dilute hydrochloric acid, dilute sodium hydroxide, cellulase, or xylanase enzymes using high-shear ultrafine grinding and high-pressure homogenization. The effect of the different pretreatments on chemical composition and structure of isolated nanofibers was studied using chemical analyses, X-ray diffraction, and Fourier transform infrared. The dimensions and properties of the isolated nanofibers were followed at the different processing stages using optical microscopy, transmission electron microscopy, atomic force microscopy, and tensile properties (wet and dry). The diameter of the microfibrils was in the range of 7-30 nm for untreated and pretreated bagasse pulps while larger microfibrillar bands (to 150 nm wide) were observed for untreated bagasse pulp than the pretreated pulps (to 90 nm wide). Nanopaper sheets made from nanofibers isolated from alkali- and xylanase-treated pulps showed better wet and dry tensile strength than those made from the other pulps.