Multidrug-resistant (MDR) Enterococcus faecalis is involved in various illnesses. This research seeks to categorize and distinguish E. faecalis in various medical tests by VITEK and 16SRNA, particularly those with vancomycin-resistance. Identification of genes those are resistant to VanA, VanB, and VanC. And also use of Chitosan L. acidophilus nanoparticles serving like a normal remedy for vancomycin-resistant MDR (VRMDR) E. faecalis strains. E. faecalis became apparent in 85/145 body fluids, stool and urine samples (58.62%). 45/85 samples were found to be VRMDR E. faecalis. The outcome demonstrated that 66.66% of E. faecalis separates possessed the VanA gene, 53.33% harbored the VanB gene and 46.66% harbored the VanC gene, while 8.88% of them harbored the three genes. The effect of Chitosan L. acidophilus nanoparticles on VRMDR E. faecalis containing the three resistance genes that 20 µg/ml was the minimal bactericidal concentration, while 10µg/ml was the minimal inhibitory concentration. Consequently, as detected by TEM, Chitosan L. acidophilus nanoparticles could affect by damaging the cell wall, nuclear material and cytoplasmic structures. The IC50 of Chitosan L. acidophilus nanoparticles were 550.02±72.9ug/ml and the imipenem drug was 301.72±4.05ug/ml, showing that imipenem was more cytotoxic on human gastric epithelial cell line (GES1) than Chitosan L. acidophilus nanoparticles.