In the present study, three different extracted nanocrystalline cellulose (NCs) with different moieties (-COO-, SO4--, and PO4---) were used as both capping and reducing agents for biogenic fabrication of nanocrystalline cellulose-silver (NC-Ag) bio-nanocomposites via a simple, easy and green process. UV-Vis absorption spectra study over a long duration of storage indicates that all colloidal suspensions of the prepared NC-Ag bio-nanocomposites were stable. Disk-diffusion agar method demonstrated various microbial inhibition regions by the three fabricated NC-Ag bio-nanocomposites against four bacterial species: Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus. The results obtained by FTIR, UV-visible, XRD, and TEM also indicate the potential effects on the physicomechanical properties of chitosan film that occur after loading it with 7% (wt/wt) NC-Ag bio-nanocomposites. Remarkably, the antimicrobial efficacy of the chitosan films differed depending on the films and microorganisms tested. Likewise, the comparison of microbial reduction rates results revealed that Gram-negative bacteria and Aspergillusniger fungus were more susceptible to NC-Ag bio-nanocomposites' microbiocidal effects. Thus, these chitosan bio-nanocomposite films can be considered to have broad-spectrum microbicidal efficacy.