(1) Background: Alkalihalobacillus clausii AO1125 is a Gram-positive, motile, spore-forming bacterium with potential as a probiotic due to its broad-spectrum antimicrobial activity, inhibiting pathogens like Listeria monocytogenes, Staphylococcus aureus, and Clostridium difficile, as well as anti-rotavirus activity. Its resilience in gastrointestinal conditions suggests benefits for gut health. This study evaluates the safety and probiotic potential of A. clausii AO1125. (2) Methods: Genome annotation identified genes linked to probiotic traits such as stress resistance, gut colonization, immune modulation, and antimicrobial production. The genome was screened for antibiotic resistance genes using CARD, bacteriocin clusters using BAGEL4, and virulence factors via VFDB. Cytotoxicity was assessed on Vero cells and erythrocytes, and a Phase I, double-blind, placebo-controlled clinical trial was conducted with 99 healthy volunteers (50 AO1125, 49 placebo). (3) Results: Genomic analysis confirmed minimal antibiotic resistance genes and the absence of virulence factors, supporting safety. A. clausii AO1125 showed no pathogenicity, cytotoxicity, or hemolytic activity and was well-tolerated in clinical settings, with mild, transient abdominal gas as the most common adverse event. (4) Conclusions: The safety profile and genetic basis for probiotic and antimicrobial properties support A. clausii AO1125 as a promising probiotic candidate for gastrointestinal health, warranting further clinical research.