Polydimethylsiloxane‐based polyurethane (PU)/crystalline nanocellulose (CNC) bionanocomposites were produced via in situ prepolymer approach. Also, hexamethylene diisocyanate as aliphatic diisocyanate monomer and 1,4‐butanediol as aliphatic chain extender were used. CNC was incorporated into PU matrix to prepare different PU/CNC bionanocomposites. Effect of CNC content on the properties of the bionanocomposites such as thermal, thermophysical, microstructure, and in vitro cytotoxicity was investigated. According to the results, incorporating CNCs into PU matrix significantly affected the hydrogen bonding between different microstructures of matrix. Adding different amounts of CNCs affected the thermal and thermophysical properties of bionanocomposites. Also, higher amounts of CNCs resulted in lower crystallization of hard segment. Neat PU matrix showed a moderate cytotoxicity behavior against human fibroblast cells. However, incorporating CNCs significantly improved the cytotoxicity behavior of bionanocomposites where by addition of 2 wt % of CNCs, cell viability increased to 90–100%. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1771–1778, 2019.