Lignocellulose is a potential raw material for film and membrane applications, such as packaging for every day consumables or a supporting barrier layer for flexible electronics. Here, lignin-containing cationic wood nanofiber (CWNF) films were produced using sawdust as the starting material. Sawdust was directly cationized using four different aqueous solvents containing tetraethylammonium hydroxide with different carbamides (urea, methylurea, ethylurea, or dimethylurea) and glycidyltrimethylammonium chloride as the cationization agent. Cationic wood was obtained with a high cationic group content (around 1.5 mmol g⁻¹) and yield (89–100%), and CWNFs were easily obtained by mechanical disintegration as a water suspension. The films produced using solvent-casting exhibited excellent visible-light transparence (around 80% at 600 nm), and the presence of lignin allowed high UV-adsorption (below 380 nm, transmittance was under 1%). The films showed high oxygen barrier properties (below 400 and 4000 m³ μm/m² day atm at a relative humidity of 50 and 92%, respectively) and good mechanical strength. An antimicrobial test conducted using the disk method showed that the CWNF films exhibited bacterial anti-adhesive properties with a small inhibition zone. CWNFs are therefore potential environmentally friendly packaging materials to prevent food spoilage, or useful as a UV-absorption layer for electronic devices such as solar cells.