Bee propolis is one of the most common natural extracts and has gained significant interest in biomedicine due to its high content of phenolic acids and flavonoids, which are responsible for the antioxidant activity of natural products. The present study report that the propolis extract (PE) was produced by ethanol in the surrounding environment. The obtained PE was added at different concentrations to cellulose nanofiber (CNF)/poly(vinyl alcohol) (PVA), and subjected to freezing thawing and freeze drying methods to develop porous bioactive matrices. Scanning electron microscope (SEM) observations displayed that the prepared samples had an interconnected porous structure with pore sizes in the range of 10–100 μm. The high performance liquid chromatography (HPLC) results of PE showed around 18 polyphenol compounds, with the highest amounts of hesperetin (183.7 µg/mL), chlorogenic acid (96.9 µg/mL) and caffeic acid (90.2 µg/mL). The antibacterial activity results indicated that both PE and PE-functionalized hydrogels exhibited a potential antimicrobial effects againstEscherichia coli,Salmonella typhimurium, Streptococcus mutans, andCandida albicans. The in vitro test cell culture experiments indicated that the cells on the PE-functionalized hydrogels had the greatest viability, adhesion, and spreading of cells. Altogether, these data highlight the interesting effect of propolis bio-functionalization to enhance the biological features of CNF/PVA hydrogel as a functional matrix for biomedical applications.