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Antimicrobial resistance has recently been considered an emerging catastrophe globally. The public health and environmental threats were aggravated by the injudicious use of antibiotics in animal farming, aquaculture, and croup fields, etc. Consequently, failure of antibiotic therapies is common because of the emergence of multidrug-resistant (MDR) bacteria in the environment. Thus, the reduction in antibiotic spillage in the environment could be an important step for overcoming this situation. Bear in mind, this research was focused on the green synthesis of chitosan nanoparticles (ChiNPs) using Citrus lemon (Assam lemon) extract as a cross-linker and application in controlling MDR bacteria to reduce the antibiotic spillage in that sector. For evaluating antibacterial activity, Staphylococcus aureus and Escherichia coli were isolated from environmental specimens, and their multidrug-resistant pattern were identified both phenotypically by disk diffusion and genotypically by detecting methicillin- (mecA), penicillin- (blaZ), and streptomycin (aadA1)-resistance encoding genes. The inhibitory zone's diameter was employed as a parameter for determining the antibacterial effect against MDR bacteria revealing 30 ± 0.4 mm, 34 ± 0.2 mm, and 36 ± 0.8 mm zones of inhibition against methicillin- (mecA) and penicillin (blaZ)-resistant S. aureus, and streptomycin (aadA1)-resistant E. coli, respectively. The minimum inhibitory concentration at 0.31 mg/mL and minimum bactericidal concentration at 0.62 mg/mL of yielded ChiNPs were used as the broad-spectrum application against MDR bacteria. Finally, the biocompatibility of ChiNPs was confirmed by showing a negligible decrease in BHK-21 cell viability at doses less than 2 MIC, suggesting their potential for future application in antibiotic-free farming practices. [ABSTRACT FROM AUTHOR]