In the current study, the effect on packaged beef fillets (1 × 5 × 8 cm) of using active chitosan film (1%) was investigated. The fillets were stored at 4 °C for 12 days, and the film contained ɛ-polylysine (ɛ-PL) (0.3, 0.6, and 0.9% w/w). Chemical, microbiological, sensory properties, and quality indices of the fillets were investigated. Added to these factors was an assessment of the influence of ɛ-polylysine incorporation on the optical, structural, barrier, and mechanical specifications (elongation at break and tensile strength) of chitosan films. Based on the findings, a significant difference among the corresponding values to thickness, color, water vapor permeability (WVP), and mechanical specifications between the treated films by ɛ-PL and untreated films were noted. In addition, higher values of thickness and tensile strength were correlated with ɛ-PL added active chitosan films while compared with control samples. Additionally, no significant differences regarding the proximate composition (including protein, moisture, and fat) among beef fillet samples were observed. In this regard, due to significantly lower levels of pH, TVB-N, and TBARS ɛ-PL in enriched films, this technique demonstrated some protective effects on beef fillets. Another observation was that lower levels of the total viable count, coliform, mold, yeasts, and higher sensory properties were significantly associated with samples with added ɛ-PL (0.9%). Therefore, adding ɛ-PL into chitosan films could be introduced as an effective technique to extend the shelf life of beef fillets and maintain their quality indices during refrigerated storage.