<p>Live biotherapeutic products are emerging as supports for host physiology and gastrointestinal health. However, despite their broad use and apparent safety, the in vivo toxicity of new probiotic strains must be rigorously evaluated before human application. <i>Bacillus safensis</i> NMCC-189, a spore-forming bacterium with promising probiotic potential, was assessed through a 14-day short-term repeated-dose tolerability study to confirm initial safety followed by sub-acute (28-day) oral toxicity study conducted in accordance with Organisation for Economic Co-operation and Development (OECD) test guideline 407. Male and female Balb/c mice were orally administered low (1 × 10⁸&#xa0;CFU/mL) and high doses (1 × 10¹⁰&#xa0;CFU/mL) of <i>B. safensis</i> NMCC-189 corresponding to approximately 1 × 10⁹&#xa0;CFU/kg/day 1 × 10¹¹&#xa0;CFU/kg/day. Dose selection was based on established probiotic safety ranges and toxicological principles. Clinical signs, survival, body weight, food intake, haematology, serum biochemistry, organ weights, and histopathology were examined, alongside daily in-life monitoring, randomisation, and blinding. No morbidity, mortality, or treatment related clinical abnormalities were observed in either study phase. Body weight gain, food consumption, and behavioural patterns remained within normal physiological limits. Haematological and biochemical parameters showed no toxicologically relevant changes, and minor fluctuations remained within reference ranges. Gross and microscopic examination of major organs revealed no pathological changes. The no observed adverse effect level (NOAEL) was 1 × 10¹¹&#xa0;CFU/kg/day. These findings suggest that <i>B. safensis</i> is well tolerated following oral administration and support its safety as a probiotic candidate. Overall, the combined study design provided a structured framework for the preclinical toxicological assessment of live biotherapeutic products.</p>