Using culture enrichment methods, 100 strains of bacilli of lactic acid bacteria (LAB) were isolated from honeybeeApis mellifera intermissaand fresh honey, collected from apiaries located in the north-east of Algeria. Amongst all of the isolated LAB, 19 selected strains were closely affiliated to four species—Fructobacillus fructosus(10),Apilactobacillus kunkeei(5),Lactobacillus kimbladiiand/orLactobacillus kullabergensis(4)—using phylogenetic and phenotypic approaches. The in vitro probiotic characteristics (simulated gastrointestinal fluids tolerance, autoaggregation and hydrophobicity abilities, antimicrobial activity and cholesterol reduction) and safety properties (hemolytic activity, antibiotic resistance and absence of biogenic amines) were evaluated. The results indicated that some strains showed promising potential probiotic properties. In addition, neither hemolytic activity nor biogenic amines were produced. The carbohydrate fermentation test (API 50 CHL) revealed that the strains could efficiently use a broad range of carbohydrates; additionally, four strains belonging toApilactobacillus kunkeeiandFructobacillus fructosuswere found to be exopolysaccharides (EPS) producers. This study demonstrates the honeybeeApis mellifera intermissaand one of her products as a reservoir for novel LAB with potential probiotic features, suggesting suitability for promoting host health.