Introduction: Although antibiotics have revolutionized health care by saving lives, the evolution of both pathogenic and commensal antibiotic-resistant bacteria are emerging as a threat in the health sector. As for Lactobacillus spp., it is usually a non-pathogenic bacteria. However, it can cause infection in immunocompromised condition. In this study, Lactobacillus spp. has been isolated from the faeces of infants with Hirschsprung disease (HD), which is congenital aganglionosis of intestine, where surgical approach and antibiotics are frequently used as medical intervention. The aim of this study is to assess the antibiotic resistance pattern and determine the presence of resistance genes, if any, in Lactobacillus spp. isolated from HD infants with ileostomy. Methodology: Six Lactobacillus spp. were isolated from faeces of six HD infants and confirmed using both conventional and molecular methods. Antibiotic resistance pattern was checked through disc diffusion method and was further investigated for the presence of antibiotic resistance genes (blaTEM, blaCTX-M, blaOXA-2, blaIMP, blaVIM-2, blaNDM-1 and mcr-1). Results: Antibiotic susceptibility of the isolates showed high level of resistance towards cephalosporins, oxacillin, aztreonam, meropenem and polymyxin group. However, four of the isolates showed the presence of blaCTX-M gene after PCR amplification. Conclusions: To our knowledge, this is the first report on the presence of antibiotic resistance gene blaCTX-M in Lactobacillus spp. and this presence may pose a serious threat in treatment regimen. As not much is known regarding the presence of blaCTX-M in Lactobacillus spp., this finding may provide new light to research on antibiotic resistance in gut microflora.