Green synthesis of nanoparticles for use in food packaging or biomedical applications is attracting increasing interest. In this study, the effect of the degree of substitution (0.7, 0.9 and 1.2) of a carboxymethylcellulose polymer matrix on the synthesis and properties of silver nanoparticles using melanin as a reductant was investigated. For this purpose, the mechanical, UV–Vis barrier, crystallinity, morphology, antioxidant and antimicrobial properties of the films were determined, as well as the color and changes in chemical bonds. The degree of substitution effected noticeable changes in the color of the films (the L* parameter was 2.87 ± 0.76, 5.59 ± 1.30 and 13.45 ± 1.11 for CMC 0.7 + Ag, CMC 0.9 + Ag and CMC 1.2 + Ag samples, respectively), the UV–Vis barrier properties (the transmittance at 280 nm was 4.51 ± 0.58, 7.65 ± 0.84 and 7.98 ± 0.75 for CMC 0.7 + Ag, CMC 0.9 + Ag and CMC 1.2 + Ag, respectively) or the antimicrobial properties of the films (the higher the degree of substitution, the better the antimicrobial properties of the silver nanoparticle-modified films). The differences in the properties of films with silver nanoparticles synthesized in situ might be linked to the increasing dispersion of silver nanoparticles as the degree of CMC substitution increases. Potentially, such films could be used in food packaging or biomedical applications.