Background: The identification of novel bacterial species from the intestines of yaks residing on the Qinghai–Tibet Plateau is pivotal in advancing our understanding of host–microbiome interactions and represents a promising avenue for microbial drug discovery. Methods: In this study, we conducted a polyphasic taxonomic analysis and bioactive assays on a Bacillus strain, designated Bos-x6-28, isolated from yak feces. Results: The findings revealed that strain Bos-x6-28 shares a high 16S rRNA gene sequence similarity (98.91%) with B. xiamenensis HYC-10T and B. zhangzhouensis DW5-4T, suggesting close phylogenetic affinity. Physiological and biochemical characterizations demonstrated that Bos-x6-28 could utilize nine carbon sources, including D-galactose, inositol, and fructose, alongside nine nitrogen sources, such as threonine, alanine, and proline. Analysis of biochemical markers indicated that Bos-x6-28’s cell wall hydrolysates contained mannose, glucose, and meso-2,6-diaminopimelic acid, while menaquinone-7 (MK-7), phosphatidylethanolamine (PE), phosphatidylcholine (PC), and phosphatidylglycerol (DPG) were found in the cell membrane. The primary cellular fatty acids included C16:0 (28.00%), cyclo-C17:0 (19.97%), C14:0 (8.75%), cyclo-C19:0 (8.52%), iso-C15:0 (5.49%), anteiso-C15:0 (4.61%), and C12:0 (3.15%). Whole-genome sequencing identified a genome size of 3.33 Mbp with 3353 coding genes. Digital DNA–DNA hybridization (dDDH) and average nucleotide identity (ANI) analyses confirmed Bos-x6-28 as a novel species, hereby named B. maqinnsis Bos-x6-28 (MCCC 1K09379). Further genomic analysis unveiled biosynthetic gene clusters encoding bioactive natural compounds, including β-lactones, sactipeptides, fengycin, and lichenysin analogs. Additionally, in vitro assays demonstrated that this strain exhibits antibacterial and cytotoxic activities. Conclusions: These findings collectively indicate the novel Bacillus species B. maqinnsis Bos-x6-28 as a promising source for novel antibiotic and antitumor agents.
