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Background Searching for a non-antibiotic therapeutic option such as probiotics is gaining momentum nowadays. We aimed to evaluate the in vitro antibacterial ability of cell-free supernatant (CFS) of selected Lactobacillus strains (with probiotic properties) against clinical isolates of OXA-48-producing multidrug-resistant (MDR) Klebsiella pneumoniae separately and in combination with cefoperazone antibiotic. Methods Over a period of 8 months, a cross-sectional experimental study involving 590 Klebsiella pneumoniae isolates was done. Our study took place at The Specialized Pediatric Teaching Hospital of Cairo University. Of the 590 Klebsiella pneumoniae isolates collected from blood cultures, pus, endotracheal aspirates, and pleural fluid, only 50 unrepeated clinical isolates of MDR Klebsiella pneumoniae-producing OXA-48-like detected by CHROMID® OXA-48 (bioMérieux, France) were selected for our study. After determining the minimal inhibitory concentration of CFS of ten Lactobacillus strains and cefoperazone each, the synergistic effect of both was tested. Results Among ten tested Lactobacillus spp., a significant increase in the mean value of inhibition zone diameter with CFS of L. helveticus (14.32 mm) and L. rhamnosus (13.3 mm) was detected separately. On the contrary, an antagonistic activity against all tested isolates was detected upon combination of Lactobacilli with cefoperazone (512 μg/ml). The mean value of inhibition zone diameter of L. helveticus CFS+ cefoperazone was (11.0 mm) and for L. rhamnosus CFS+ cefoperazone was (10.88 mm) (p value <0.001). Conclusion The antimicrobial efficiency of using CFS of Lactobacillus species separately indicates that these therapies may be a substitute treatment strategy against MDR Klebsiella pneumoniae.