Bacterial biofilms gained attention in the medical field with the emergence of antibiotic-resistant strains. The potency of lactic acid bacteria (LAB), isolated from kefir, against the development of a biofilm populated by the multidrug-resistant (MDR) Klebsiella pneumoniae was accessed in this study. The ability of LAB to inhibit biofilm formation was screened using the crystal violet method, and the morphology of the biofilms was analyzed using electron microscopy. Cell viability of K. pneumoniae after administration of LAB cell free supernatant was determined by MTT assay. Lactobacillus isolates were characterized using partial sequencing against 16S rRNA. Quorum sensing inhibition of K. pneumoniae by LAB was conducted with the help of the AHL reporter strain. Result showed that addition of cell free supernatant (CFS) as well as pellet of 40 isolates LAB on to K. pneumoniae culture could decrease their culture absorbance. Among those isolates, LAB G24 and G25 from grain kefir and LAB K10 and K15 from milk kefir demonstrated the strongest effect by reducing the absorbance by eight to nine-fold. Analysis of biofilm morphology showed that the CFS of Lactobacillus could prevent the attachment between K. pneumoniae cells. In addition, cell viability of K. pneumoniae was significantly reduced after the addition of the LABs CFS. Partial sequencing of 16S rRNA identified the LAB isolate as Lactobacillus helveticus. In conclusion the L. helveticus isolated from kefir demonstrated anti-biofilm activity against MDR K. pneumoniae. L. helveticus are potential probiotic strains for therapeutic application in the clearance of MDR K. pneumoniae.