Aims: This study was aimed to produce biodegradable cellulose acetate films impregnated with bacteriocin-like inhibitory substances to be used in food packaging. Methodology and results: Bacterial isolates were isolated from different sources and tested for their antimicrobial activity by agar well diffusion assay. The isolate that showed the highest antimicrobial activity against Staphylococcus aureus ATCC 29213, Escherichia coli ATCC 51659, Pseudomonas aeruginosa MG847103 and Streptococcus mutans ATCC 25175 was identified as Lactiplantibacillus plantarum strain N1 (OM019104) based on 16S rRNA based method of identification. The extracted bacteriocin-like inhibitory substances were partially purified with acetone precipitation and SP-Sephroase cation exchange chromatography. There was no change in the antimicrobial activity after treatment with the catalase enzyme, but there was a total loss in the activity after treatment with proteolytic enzymes. The obtained bacteriocin-like inhibitory substances showed pH stability over a wide range of pH values and thermal stability as it recovered 95% of its antimicrobial activity even after autoclaving for 15 min. Different concentrations of cellulose acetate (3 to 12%) were tested to develop the active antimicrobial films. The most flexible concentrations for food packaging were prepared and impregnated with partially purified bacteriocin-like inhibitory substances. The prepared films showed promising antimicrobial activity against S. aureus. Conclusion, significance and impact of study This study highlights the usage of active packaging in food preservation. Cellulose acetate films activated with the partially purified bacteriocin-like inhibitory substances have anti-staphylococcal activity, which can potentially be used in food packaging to prolong the shelf-life of perishable foo