The aim of this study was to screen intestinal lactobacilli strains for their advantageous properties to select those that could be used for the development of novel gastrointestinal probiotics. Ninety-three isolates were subjected to screening procedures. Fifty-nine percent of the examined lactobacilli showed the ability to auto-aggregate, 97% tolerated a high concentration of bile (2% w/v), 50% survived for 4 h at pH 3.0, and all strains were unaffected by a high concentration of pancreatin (0.5% w/v). One Lactobacillus buchneri strain was resistant to tetracycline. None of the tested strains caused lysis of human erythrocytes. Six potential probiotic strains were selected for safety evaluation in a mouse model. Five of 6 strains caused no translocation, and were considered safe. In conclusion, several strains belonging to different species and fermentation groups were found that have properties required for a potential probiotic strain. This study was the first phase of a multi-phase study aimed to develop a novel, safe and efficient prophylactic and therapeutic treatment system against gastrointestinal infections using genetically modified probiotic lactobacilli.