Nanofibers (NF) were isolated from bleached bagasse and rice straw pulps. The pulps were refined using high-shear ultrafine grinder and then homogenized using high-pressure homogenizer. The efficiency of the used isolation processes was studied by optical microscopy (OM), transmission electron microscopy (TEM), atomic force microscopy (AFM), and testing the tensile properties (wet and dry) of nanopaper sheets made from the nanofibers. In addition, opacity and porosity of nanopaper sheets made after different processing steps were investigated. The microscopy studies showed that the processes used resulted in nanofibers with diameters ranging from 3.5 to 60 nm. The results indicated that main isolation of nanofibers took place during refining using the ultrafine grinding process, while high-pressure homogenization resulted in smaller and more homogeneous size of nanofibers. Nanopaper sheets made from bagasse showed better wet and dry tensile strength properties than those made of rice straw