The safety profile of <i>Lentilactobacillus buchneri</i> KU200793, which has neuroprotective effects, was comprehensively evaluated through both phenotypic and genotypic analyses. Phenotypically, the strain exhibited no β-hemolysis, mucin degradation, indole production, gelatin liquefaction, urease activity, or β-glucuronidase activity. Additionally, it did not produce D-lactate, and only trace amounts of spermidine were detected among the biogenic amines. Furthermore, <i>L. buchneri</i> KU200793 did not exhibit bile salt deconjugation, further supporting its safety profile. However, its tetracycline resistance exceeded the threshold set by the European Food Safety Authority. Genotypic analysis using the HGTree program identified <i>tetA</i>(<i>58</i>) and <i>nalD</i> genes with sequence similarities of 33.64% and 30.17%, respectively, indicating a low level of homology. These findings suggest that tetracycline resistance in <i>L. buchneri</i> KU200793 is unlikely to have been acquired through horizontal gene transfer, thereby minimizing the risk of resistance gene dissemination. This study underscores the importance of comprehensive safety assessments to evaluate the suitability of <i>L. buchneri</i> KU200793 for probiotic applications.