The aim of this study was to evaluate the antifungal activity of Thymus vulgaris essential oil (TEO) against fungal isolates from cheese and its potential as an active component in cellulose acetate films for application on cheese, and to perform the characterization of the films. The cheese rind isolates were identified as Penicillium crustosum QCP1 and Aspergillus flavus QCA2. TEO exhibited significant in vitro inhibitory activity against all isolates, with inhibition zones exceeding 40.0 mm and minimum inhibitory concentrations (MICs) of 1.0 µL mL⁻¹ and 0.8 µL mL⁻¹ for P. crustosum QCP1 and A. flavus QCA2, respectively. Incorporating TEO into cellulose acetate films altered the film's physical properties, particularly morphology and elongation. The control film, composed solely of acetate, showed the lowest values for most properties evaluated, except for tensile strength (MPa) (38.89 ± 5.29). For water permeability and water vapor permeability, no significant differences were observed between the control film and the active film containing TEO at tenfold MFC concentrations for each isolate. In in vitro tests with the active film, P. crustosum QCP1 demonstrated higher sensitivity
however, the active film effectively inhibited the growth of both fungal isolates on cheese slices for 30 days. It was concluded that TEO has antifungal potential against isolates in more than one type of methodology and, when added to cellulose acetate film, was effective in controlling fungal mycelial growth, both in vitro and in situ, extending shelf life by up to 2 times, indicating a promising application as active packaging in cheeses.