As demand on the safety and quality of the product in food packaging is increased, nanocellulose based transparent paper with resistance against microbial activity becomes more important. In this study, transparent cellulose film was prepared from ginger nanofiber with chemicals and ultrasonication. After purification using acid hydrolysis, ginger fiber had high cellulose content (88%). Ultrasonication of the suspension of the chemically treated fibers for 1 h decreased their diameter to 54.3 nm. Drying of the nano-sized fiber suspension resulted in a transparent film with 5 μm thickness. This film transmitted 83.3% of the light at 650 nm and showed antimicrobial activity. It had high thermal stability with the maximum decomposition temperature peak at 353 °C. This was 17% higher than that of raw ginger fiber (302 °C). The transparent film had a crystallinity index of 48%, and lower moisture resistance than film from chemically treated cellulose fiber. This study promoted transparent nanocellulose film with good antimicrobial properties from abundant and available ginger fibers. Drying of the nano-sized fiber suspension resulted in an optically transparent film showing antimicrobial activity. Display omitted •Ginger nanofibers were prepared with chemical treatment and ultrasonication.•Drying of the nano-sized fiber suspension resulted in an optically transparent film.•This transparent film had high thermal stability and showed antimicrobial activity.