© 2022, The Author(s), under exclusive licence to Springer Nature B.V.Currently, researchers are focusing on the development of nano-additive preservatives during the worldwide COVID-19 pandemic. This research aimed to constitute a small sized preservative nano-formulation which emerges from the biopolymer carboxymethyl cellulose (a green stabilizing agent) and hydromagnesite stromatolite (a fossilized natural additive). In this study, we investigated the optimization of the experimental design of carboxymethyl cellulose/hydromagnesite stromatolite (CMC/HS) bio-nanocomposites using a green and one-step sonochemical method at room temperature. In addition, we constructed a mathematical model which relates the intrinsic viscosity with all operating variables, and we carried out statistical error analysis to assess the validity of the proposed model. The characterization and chemical functional groups of CMC/HS bio-nanocomposites were determined by different advanced techniques such as SEM, HRTEM, DLS, FTIR, XRD, and BET. The challenge test was used to show the preservative efficacy of CMC/HS bio-nanocomposites against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Candida albicans, and Aspergillus brasiliensis. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltrazolium bromide (MTT) assay was performed on L929 cells to evaluate the in vitro cytotoxicity of CMC/HS bio-nanocomposites. According to the results, we showed that the synthesized CMC/HS bio-nanocomposites have no cytototoxic effects on L929 fibroblast cells and could be considered to be an alternative green nano-additive preservative against pathogenic microorganisms.