In the present study, 20 selected Lactobacillus strains already characterized in a previous research for their capability to grow in conditions simulating the intestinal environment, their resistance to antibiotics, their antibacterial activity and their adhesion capability to intestinal human Caco-2 TC7 and HT-29 MTX cell lines, were further investigated to explore more their probiotic properties. Growth behaviour in the presence of prebiotic (fructooligosaccharides (FOS) and lactulose) at a concentration of 2%, cholesterol removal by measuring the residual cholesterol in medium supplemented with cholesterol, ability to deconjugate bile salts using BSH enzyme and antioxidant activity of culture supernatant of Lactobacillus strains by ABTS .+ and DPPH methods were analyzed. All probiotic strains demonstrated important prebiotic assimilation (P > 0.05) even with OD 600 > 3 after 30 h of contact, cholesterol removal ability with maximum percentage of 57% after 24 h of contact and they were found to liberate significantly (P < 0.05) more cholic acid with maximum of 0.40 mM of sodium glycocholate, 0.33 mM of sodium taurocholate and 0.41 mM of their mixte and scavenge both radicals with 52% and 2.19% of ABTS .+ and DPPH respectively. This study confirmed the suitability of these probiotic strains for application in functional food formulations especially where cholesterol reduction and antioxidant activity in food are needed to assess possible in vivo human health benefits.