The purpose of this study is to investigate a new applicable and promising treatment for American Foulbrood (AFB) disease in honeybee colonies. AFB is a very serious honeybee disease caused by the spore-forming bacteria Paenibacillus larvae larvae (P. l. larvae). Five honeybee endogenous lactic acid bacteria (LAB), isolated from honeybee workers' guts, were previously identified and tested for their inhibitory effects on P. l. larva in vitro. Artificial infection was accompanied by the administration of a mixture of the five LAB strains, two belong to Lactobacillus plantarum, two different strains of Lactobacillus kunkeei, one strain of Lactobacillus sp..  It was observed that the honeybee endogenous LABs inhibited P. l. larvae in an in vivo system. LAB mixture added to the larval food in honeybee colonies significantly reduced the number of infected larvae (P ≃ 0.000, p < 0.001). Confidence intervals analysis showed no significant difference from adding LAB to the food on first or second-day post infection and throughout the feeding period. In vivo studies demonstrated that LAB microbiota in Apis mellifera inhibits the bacterial brood pathogen P. l. larvae. The results pointed to new avenues for the prophylactic or therapeutic treatment of honeybee diseases.