Nanofibers isolated from unbleached neutral sulfite rice straw pulp were used to prepare transparent films without the need to modify the isolated rice straw nanofibers (RSNF). RSNF with loading from 1.25 to 10 wt.% were mixed with cellulose acetate (CA) solution in acetone and films were formed by casting. The films were characterized regarding their transparency and light transmittance, microstructure, mechanical properties, crystallinity, water contact angle, porosity, water vapor permeability, and thermal properties. The results showed good dispersion of RSNF in CA matrix and films with good transparency and homogeneity could be prepared at RSNF loadings of less than 5%. As shown from contact angle and atomic force microscopy (AFM) measurements, the RSNF resulted in increased hydrophilic nature and roughness of the films. No significant improvement in tensile strength and Young’s modulus was recorded as a result of adding RSNF to CA. Addition of the RSNF did not significantly affect the porosity, crystallinity and melting temperature of CA, but slightly increased its glass transition temperature