Background: Fructophilic lactic acid bacteria (FLAB) are a group of LAB with unique growth features and are regarded as potential bioactive compound manufacturers. FLAB is discovered in D-fructose rich niches that favors D-fructose as a growth substrate triumphs over D-glucose. On D-glucose, they require electron acceptors to proliferate. The organisms have similar metabolic processes for carbohydrates. Fructobacillus spp. are Lactobacillus kunkeei, are members of this unique group. Methodology: Inhibitory effect of filtrate from four FLAB isolates from honeybee was detected against the growth of pathogenic Klebsiella pneumoniae isolates. Biofilm formation, and the expression of fimH, mrkD, and NDM-1, and OXA-1 virulence genes were also tested. Results: It was found that FLAB has antibacterial and antibiofilm effect. The incubation of K. pneumoniae with FLAB suspension at different three-time intervals (3, 6, and 9 hrs.) decreased expression of fimH, mrkD, NDM-1, and OXA-1 virulence genes compared with the expression of the same genes in the absence of FLAB. Conclusion: all FLAB filtrate isolates tested, K. pneumoniae fimH, mrkD, NDM-1, and OXA-1 virulence gene expression was down-regulated, when compared to the control culture.