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Environmental pollution and food safety are both issues of global concern. In this sense, sustainable and antimicrobial nanocomposites based on cellulose/poly (vinyl alcohol) blend incorporated with natamycin and cellulose nanocrystals (CNC) were manufactured and characterized. The developed films were evaluated according to their mechanical and optical properties, and their barrier to oxygen and water vapor permeation. The antimycotic activity was evaluated in vitro against fungi and yeasts. The film's potential to act as an active packaging for Minas cheese preservation was also assessed. The incorporation of CNC increased the films' tensile strength; however, it did not influence the barrier properties to water vapor (4.12 × 10−7 g·cm.m−1·h−1·Pa−1) and oxygen (3.64 × 10−13 g·cm·m−1·h−1·Pa−1). The incorporation of natamycin, on the other hand, resulted in films that were more opaque (around 24%) and of a yellowish color. The active nanocomposites developed showed antimicrobial effects against all analyzed fungi and yeasts (approximately 35 mm of inhibition zone) and were able to control the growth of S. cerevisiae in cheese, reducing a log cycle until the 12th day of storage. Since they performed well in vitro and on food, it was concluded that the films showed potential to be applied in Minas cheese preservation. [ABSTRACT FROM AUTHOR]