Human normal flora is a source of probiotics. The safety characteristics of a specific isolate determine its application in foods or drugs. The food-borne-pathogen antagonist strain Lactobacillus gasseri HMV18 is one of the isolates from normal human flora. In this work, we assessed the in vitro pH tolerance, bile tolerance, biogenic amine production, mucin utilization, and safety of in vivo administration to mice to evaluate general health, organ-body weight index, organ histopathological change, whether L. gasseri HMV18 can colonize in the gut or modulate the gut microbiota after oral administration. The results suggest that L. gasseri HMV18 can tolerate pH 3 for 2 h, 3% bile for 3 h, biogenic amine negative, mucin usage negative, does not encode verified toxins, and cause no visible change in mice’s organs. L. gasseri HMV18 might not colonize in mice’s gut, but can significantly affect the structure of gut microbiota. A bibliographical survey suggested that there were as few as 8 opportunistic infection cases from 1984 to 2022 and that the possibility for L. gasseri to cause infection is relatively low. Therefore, this work provides a basis for the foods or drugs application of L. gasseri HMV18 and gives a map of experiments for the safety assessment of probiotics.