The current study aims to prepare carbohydrate nanocomposite based on 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) oxidized cellulose nanofibers (CNFs) /chitosan chloride (Ch) decorated with silver nanoparticles (Ag-NPs) and designed as (TEMPO/CNF/Ch/Ag-NPs) that was investigated as antimicrobial materials and applied as efficient sorbent. CNF was prepared from cellulose through TEMPO oxidation-mechanical defibrillation technique. Anionic CNF and Ch were ironically interacted to prepare novel sustainable ionic nanofibers films. Ag-NPs having approximately 20 nm were prepared in situ to decorate the ionic fibers. The effects of experimental parameters including media pH (3.0–8.0), contact time (10–120 min), and initial methylene blue (MB) concentration (50–800 ppm) on the adsorption behavior of MB onto nanocomposite films were evaluated. The results exhibited similar and fast adsorption kinetic, as illustrated by the pseudo-second-order model. The adsorption results were best suited by the Langmuir isotherm model, and the maximum adsorption capacity for the TEMPO/CNF/Ch and TEMPO/CNF/Ch/Ag-NPs nanocomposite was 369 and 432 mg/g, respectively, at the optimized media pH of 7 with a contact time of 60 min at 88 to 90 mg/g of MB. In kinetic studies, the pseudo-second-order model for nanocomposite films provided the best fit (R2 = 0.98), than pseudo-first-order model (R2 = 0.82 and 0.77). The TEMPO/CNF/Ch and TEMPO/CNF/Ch/Ag-NPs nanocomposite retains 73 and 69% of its initial adsorption performance towards MB after five consecutive runs. The formed nanocomposite showed excellent efficacy against Escherichia coli (14.3 ± 0.57 mm), followed by Candida albicans (12 ± 1 mm), Staphylococcus aureus (11.3 ± 1.15 mm), and Streptococcus mutans (10.6 ± 1.15 mm), and their antimicrobial activity was enhanced by the incorporation of Ag-NPs. The formed nanocomposite might find applications in industrial and biomedical fields.