Cellulose nanofibrils (CNFs) and silver nanoparticles (AgNPs) are nano-sized fillers widely used for enhancing the active functions of food packaging materials. However, nanomaterials may pose unexpected toxic effects on humans. Thus, the objective of this work was to develop a novel CNF/AgNP composite and investigate its properties and cytotoxicity. The CNF/AgNP composite was prepared via a reduction method using NaBH. The AgNPs embedded in the composite showed an average size of 10.72 ± 4.96 nm and a surface plasmon resonance (SPR) absorption peak at 397 nm. AgNPs were distributed individually in the composite after synthesis, but aggregated during film preparation. The formation of AgNPs disrupted hydrogen bonds between the hydroxyl groups of cellulose, weakening the hydrogen-bond intensity, as shown by FTIR. Silver ions were efficiently released from the composite film in the first 24 h. The CNF/AgNP composite exhibited inhibitory effects on Escherichia coli O157:H7 and Listeria monocytogenes. The composite (50-1000 μg/mL) did not significantly reduce the viability of Caco-2 and FHC colon cells, although the uptake of AgNPs through an endosomal mechanism was observed. These results suggest that the as-prepared CNF/AgNP composite could potentially be used as an antimicrobial material in active food packaging systems.