Recently, the improper use of antibiotics led to the development of multidrug resistance in microbial flora raising a global public health. We evaluated the antimicrobial activity of Chitosan Oligosaccharide (OCHT) against E. coil isolates during the 10 generation and ampicillin resistance for E. coil. The OCHT inhibited the growth of E. coli NCTC 9001 and isolates (E. coli O157:K88) from pig feces at a concentration of 0.001% (w/v) in 10 min by the 10 generation. Also, the E. coli showed a significant antibacterial activity at a concentration of OCHT 0.001~0.5% (w/v) in 30 min. Interestingly, OCHT showed antibacterial effect from ampicillin resistance for E. coli by plasmid DNA. Consequently, we confirmed that OCHT significantly inhibited multidrug resistance E. coli and might be selected as a more effective natural antibiotics for livestock.