A Gram-positive, rod-shaped, endospore-forming Bacillus isolate, Bi.FFUP1T, recovered in Portugal from a health product was subjected to a polyphasic study and compared with the type strains of Bacillus pumilus, Bacillus safensis, Bacillus altitudinis and Bacillus xiamenensis, the phenotypically and genotypically most closely related species. Acid production from cellobiose, Dglucose and D-mannose and absence of acid production from D-arabinose, erythritol, inositol, maltose, mannitol, raffinose, rhamnose, sorbitol, starch and L-tryptophan discriminated this new isolate from the type strains of the most closely related species. Additionally, a significant different protein and carbohydrate signature was evidenced by spectroscopic techniques, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and Fourier transform IR spectroscopy with attenuated total reflectance. Using a chemometric approach, the score plot generated by principal component analysis clearly delineated the isolate as a separate cluster. The quinone system for strain Bi.FFUP1Tcomprised predominantly menaquinone MK-7 and major polar lipids were diphosphatidylglycerol, an unidentified phospholipid and an unidentified glycolipid. Strain Bi.FFUP1Tshowed ≥99% 16S rRNA gene sequence similarity to B. safensis FO-036bT, B. pumilus (7061Tand SAFR-032), B. altitudinis 41KF2bTand B. xiamenensis HYC-10T. Differences in strain Bi.FFUP1TgyrB and rpoB sequences in comparison with the most closely related species and DNA-DNA hybridization experiments with Bi.FFUP1Tand B. pumilus ATCC 7061T, B. safensis FO-036bT, B. altitudinis 41KF2bTand B. xiamenensis HYC-10Tgave relatedness values of 39.6% (reciprocal 38.0 %), 49.9% (reciprocal 42.9 %), 61.9% (reciprocal 52.2 %) and 61.7% (reciprocal 49.2 %), respectively, supported the delineation of strain Bi.FFUP1Tas a representative of a novel species of the genus Bacillus, for which the name Bacillus invictae sp. nov. is proposed, with strain Bi.FFUP1T(=DSM 26896T=CCUG 64113T) as the type strain.