This study assessed the impact of calcium alginate (CA) film embedded with ε-polylysine nanoparticles (ε-PL-NPs) at concentrations of 500 and 1000 mg/L, or nisin nanoparticles (Ni-NPs) at 100 and 200 mg/L, both combined with beetroot extract (3 g/hg), on beef sausage quality. Compared to standard CA films, the active CA films with ε-PL-NPs demonstrated enhanced flexibility. The addition of Ni-NPs and ε-PL-NPs notably improved the CA films' barrier characteristics, with a reduction in water vapor permeability observed as nanoparticle concentration increased. CA films with ε-PL-NPs exhibited a more potent antimicrobial effect, effectively inhibiting a range of microbial growth, including total viable count, coliforms, Staphylococcus aureus, Clostridium perfringens, molds, and yeasts in beef sausages. Sausages packaged in CA films with ε-PL-NPs displayed reduced lipid oxidation and higher total phenolic content than those treated with Ni-NPs and the control samples. Consequently, CA films integrated with ε-PL-NPs at 1000 mg/L demonstrated promising capabilities in maintaining sausage quality, extending shelf life by 25%, and emerged as a novel packaging option for beef sausage with added beetroot extract. This innovative packaging approach also effectively decreased sausage nitrite levels, thereby addressing health concerns associated with nitrite consumption.