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The widespread use of conventional plastic mulch films in agriculture contributes significantly to soil pollution due to their non-biodegradable nature. This study explores the potential of novel bio-based mulch films composed of chitosan, carboxymethyl cellulose, and sodium alginate, formulated in different ratios (1:1 and 17:3), with or without enrichment with monoammonium phosphate (MAP), to serve as biodegradable films with potential nutrient-releasing functionality as alternatives to conventional plastics. A multi-analytical approach, including elemental and isotopic analysis (EA-IRMS), biodegradation assays, and pyrolysis–gas chromatography–mass spectrometry (Py-GC-MS), was employed to assess their chemical properties, degradation behavior, and environmental compatibility. The results demonstrated that the 1:1 films, both with and without MAP, achieved over 90% biodegradation within 120 days under controlled soil conditions, in agreement with international criteria for soil biodegradability. In contrast, the 17:3 films showed reduced degradation, especially without MAP enrichment, highlighting the influence of polymer composition on microbial degradation. Isotopic tracing confirmed MAP integration and revealed composition-dependent fractionation effects. Py-GC-MS provided structural fingerprints of film components and putatively annotated nitrogen-containing compounds indicative of chitosan presence. Overall, these results demonstrate that the 1:1 films can be considered viable, multifunctional, and soil-friendly alternatives to conventional plastic mulches for sustainable agriculture. Full article