This study aimed to determine the effect of Coleus aromaticus essential oil (CAEO) on the properties of an active film based on polylactic acid (PLA), chitosan (Cs), and cellulose nanocrystal (CNC) in fruit packaging. The films were fabricated via solvent casting technique. The obtained films’ antimicrobial, antioxidant, microstructural, and mechanical properties were studied. Mechanical properties state how adding essentials oil into film improves the elongation breaks significantly (p < 0.05) by 5.3 and 6.1%, respectively, with the addition of 0.4 wt% and 0.8 wt% CAEO which reflets its flexibility. The antioxidant activity of biopolymer film increased significantly (p < 0.05), with antioxidant values ranging from 6.50 to 57.50% with the elevation of CAEO. The inhibitory impact of the film against pathogenic fungus was evaluated in vitro and in vivo by comparing the film with different concentrations of essential oil (EO), as well as the control and chemical fungicide. Disc diffusion was utilised to test mycelial growth suppression, and the film containing 1.2 wt% EO produced the best results. The biofilm containing 1.2 wt% EO successfully reduced illness incidence in vivo with damaged mango. Scanning Electron Microscopy and Transmission Electron Microscopy imaging were used to observe the incorporation of CAEO in the matrix of the film. All Fourier-Transform Infrared spectra of PLA/Cs/CNC and EO blends exhibited the characteristic bands of PLA-based materials. The results indicate that the PLA/Cs/CNC/CAEO films provide a new way to improve microbial safety and extend the shelf life of mango fruit and have the potential for replacement of petroleum-based plastic for fruit packaging applications at the industrial level.