Probiotics are live microbes that give many health benefits to human beings and animals, the most studied and commonly used probiotics are Gram-positive bacteria; lactobacilli and bifidobacteria. At nowadays, Lactobacillus spp. constitute more than two-thirds of the total numbers of probiotic species. The present study aimed to characterize Lactobacillus that locally isolated from human mouth and feces as probiotics. A total of three Lactobacillus isolates; Lactobacillus fermentum Lb2, Lactobacillus rhamnosus Lb9, and Lactobacillus paracasei Lb10 were investigated in respect to acid and bile salts tolerance, antibiotics susceptibility, and cell surface hydrophobicity in vitro using bacterial adhesion to hydrocarbons method. In comparison with the other two isolates, the isolate L. fermentum Lb2 was able to grow in all pH values and in the presence of different concentrations of bile salts. Antibiotics susceptibility profile showed that the tested Lactobacillus isolates were sensitive to ampicillin, amoxicillin, and erythromycin, while they were resistant to the other antibiotics that used in this study. L. fermentum Lb2 exhibited high surface hydrophobicity (77.26%), while the other tested isolates; L. rhamnosus Lb9 and L. paracasei Lb10 revealed moderate adhesion abilities, 68.56% and 65%, respectively. L. fermentum Lb2 exhibited good probiotic behavior with respect to acid and bile salt tolerance as well as adhesion ability to hydrocarbons.