L. monocytogenes is a Gram-positive bacterial pathogen, known to cause food poisoning and systemic disease, specifically listeriosis. This species has shown resistance to many commonly used antibiotics, making the search for new alternative therapies is a pressing matter. A facile and eco-friendly sono-co-method was developed to produce Ag nanoparticles from palm sheath fiber agricultural waste, using carboxylated cellulose nanocrystals (CCNs). Spectroscopic analysis, including UV-visible, TEM, FTIR, and EDS, confirmed the successful synthesis of the CCN-Ag nano-biohybrids. The nano-biohybrids exhibited potent antibacterial activity against various L.monocytogenes strains, with inhibition zones ranging from 16 to 19 mm. Concentrations of the CCN-Ag suspension between 0.25 and 1 μg/mL were found to completely prevent the growth of L.monocytogenes. Conventional PCR analysis revealed the presence of several virulence genes, including actA, inlA, inlB, plcA, iap, and hlyA, in all the tested strains. Notably, CCN-Ag treatment significantly downregulated these genes, indicating a reduction in virulence and potential for biocontrol applications. The novelty of this research lies in the development of a sustainable and eco-friendly method for producing potent antimicrobial nanohybrids from agricultural waste. These nanohybrids' ability to effectively inhibit L.monocytogenes' growth and downregulate its virulence genes offers a promising avenue for combating this pathogenic bacterium.