Beneficial bacteria such as Lactobacillus spp. may exhibit promising therapeutic potential due to their inhibitory effects against antibiotic-resistant microorganisms. The present study aimed to evaluate the potency of Lactobacillus helveticus C2 isolated from kefir against multidrug resistant Klebsiella pneumoniae. The ability of L. helveticus C2 to inhibit MDR K. pneumoniae adhesion to intestinal enterocytes was observed in vitro. Intestinal colonization by MDR K. pneumoniae in the presence of L. helveticus C2 was further evaluated using a mice model. L. helveticus C2 exhibited strong antibacterial activity against MDR K. pneumoniae. Direct antagonism between L. helveticus C2 and MDR K. pneumoniae cells could not be ascertained in vitro. However, the adhesion of MDR K. pneumoniae to the small intestinal enterocytes of mice was inhibited by pili and outer membrane protein of L. helveticus C2. Moreover, L. helveticus C2 demonstrated inhibitory activity against MDR K. pneumoniae colonization by reducing the number of MDR K. pneumoniae colonies in BALB/c mice as induced by pellets and cell-free supernatant of L. helveticus C2. L. helveticus C2 is a probiotic strain of significant therapeutic or preventive potential due to its strong antibacterial activity, as well as the ability to inhibit the adhesion and colonization of MDR K. pneumoniae.