Chitin nanofibers (ChNFs) and cellulose nanocrystals (CNCs) have been proposed as materials for renewable packaging with low O2 transmission that protect food, medicine, and electronics. A challenge in biomass-derived functional materials is tuning both barrier and mechanical properties, while minimizing process steps. A concept that merits additional study in this field is tuning of the barrier and mechanical properties by use of oppositely charged biomass-derived fibers, through interactions that support dense film formation. We report free-standing films formed by solution casting of blends of aqueous suspensions of CNCs and ChNFs with either low degree of acetylation (LChNFs, higher charge) or high degree of acetylation (HChNFs, lower charge). While neat CNC films had the highest O2 permeability (OP), the OP was lowered by 91% by addition of at least 25 wt % LChNFs to CNCs to an OP value near 1.7 cm3 μm/m2/d/kPa. Interestingly, blends of CNCs with less highly charged, larger HChNFs had equivalently lower OP as with LChNFs. The tensile strength and strain at break of blended ChNF/CNC films was optimal compared to neat cellulose or chitin when at least 50 wt % LChNFs or HChNFs were blended with CNCs. We show that the ability to tune properties of ChNF/CNC blends was coincident with the formation of aggregates of chitin and cellulose nanomaterials, which appear to support formation of dense layers of tortuous fiber networks.