Background The health-promoting effects along with global economic importance of consuming food products supplemented with probiotic microorganisms encouraged the researchers to discover new probiotics. Results Fourteen lactic acid bacterial isolates were identified as Enterococcus mediterraneensis, Lactobacillus fermentum, and Streptococcus lutetiensis by 16S rRNA gene sequencing, and in vitro characterized for their actual probiotic potential. All E. mediterraneensis isolates were resistant to clindamycin, whereas Lb. fermentum isolates were resistant to ampicillin, clindamycin, and vancomycin. The E. mediterraneensis and Lb. fermentum isolates displayed high overall digestive survival, ranged from 1.35 ± 0.06 to 32.73 ± 0.84% and from 2.01 ± 0.01 to 23.9 ± 1.85%, respectively. All isolates displayed cell surface hydrophobicity, ranged between 15.44 ± 6.72 and 39.79 ± 2.87%. The strongest auto-aggregation capability, higher than 40%, was observed for most E. mediterraneensis and Lb. fermentum isolates. The E. mediterraneensis isolates (L2, L12, and L15), Lb. fermentum (L8, L9, and L10), and Strep. lutetiensis (L14) exhibited the greatest co-aggregation with Salmonella typhimurium, Escherichia coli O157:H7, Staphylococcus aureus, and Bacillus cereus. Fifty-seven and fourteen hundredth percent of E. mediterraneensis isolates could be considered bacteriocinogenic against E. coli O157:H7, B. cereus, and S. aureus. Conclusion This study is the first one to isolate Enterococcus mediterraneensis in Egypt and to characterize it as new species of probiotics globally. According to the results, E. mediterraneensis (L2, L12, and L15), Lb. fermentum (L8, L9, and L10), and Strep. lutetiensis (L14) are the most promising in vitro probiotic candidates.