Kemampuan probiotik dari bakteri bersifat spesifik tergantung strain. Bakteri dari genus Bacillus diketahui memiliki potensi sebagai probiotik selain bakteri asam laktat. Bacillus sp. tergolong sebagai bakteri Gram-positif dan dapat menghasilkan spora. Bacillus sp. mampu bertahan pada kondisi yang kurang menguntungkan dan menghasilkan enzim pencerna, senyawa antimikroba, dan vitamin. Penelitian ini bertujuan menganalisis potensi bakteri Bacillus subtilis SM10.1 koleksi Laboratorium Biologi UPH sebagai0l probiotik. Pengujian karakteristik probiotik yang dilakukan adalah uji toleransi terhadap pH rendah dan garam empedu, uji aktivitas antimikroba, uji kerentanan terhadap antibiotik, uji autoagregasi dan koagregasi, serta uji kemampuan adhesi terhadap sel epitel. Spora B. subtilis SM10.1 menunjukkan toleransi terhadap pH 2. Sel vegetatif B. subtilis SM10.1 menunjukkan toleransi terhadap garam empedu 0,3% dan 0,5%. Selain itu, B. subtilis SM10.1 menunjukkan aktivitas antimikroba terhadap Escherichia coli dan Staphylococcus aureus, kemampuan autoagregasi dan koagregasi dengan E. coli dan S. aureus, serta kemampuan adhesi pada sel epitel pipi. B. subtilis SM10.1 resistan terhadap antibiotik streptomycin, bacitracin, dan clindamycin. Hasil penelitian saat ini mendukung B. subtilis SM10.1 berpotensi sebagai probiotik, namun penelitian lebih lanjut diperlukan untuk mengkonfirmasi keamanan dan efek B. subtilis SM10.1 terhadap kesehatan. / Probiotic traits of bacteria are strain-specific. In addition to the known lactic acid bacteria, several bacteria of the genus Bacillus has been known to have probiotic traits. Bacillus sp. is Gram-positive and able to produce spore. Bacillus sp. can survive harsh condition and produce several beneficial metabolites, include enzymes, antimicrobial, and vitamins. This study aims to analyze the potential of UPH Biology Laboratorium collection, that is Bacillus subtilis SM10.1, as probiotic. Several tests were done to evaluate probiotic traits, include tolerance to acid and bile salts test, antimicrobial test, antibiotic susceptibility test, autoaggregation and coaggregation test, and adhesion ability to epithelial. Spore of B. subtilis SM10.1 showed tolerance to pH 2. Vegetative cell of B. subtilis SM10.1 showed tolerance to bile salts of 0.3% and 0.5% concentration. In addition, B. subtilis SM10.1 also exhibited antimicrobial activity against Escherichia coli and Staphylococcus aureus, autoaggregation and coaggregation ability with E. coli dan S. aureus, and adhesion ability to buccal epithelial cell. B. subtilis SM10.1 is resistant to antibiotics streptomycin, bacitracin, and clindamycin. Result of this study suggests B. subtilis SM10.1 has the potential to be probiotic, however further study is necessary to confirm B. subtilis SM10.1 traits and safety as probiotic.