2,3-Butanediol (2,3-BD) is a valuable platform chemical with extensive industrial applications. The demand for its safe and economic microbial synthesis resulted in increased interest in the isolation of non-pathogenic producers capable of converting cheap and renewable materials. This study reports the isolation of 62 new non-pathogenic Bacillus strains producing 2,3-BD. Three strains were found, including Bacillus velezensis 5RB, B. toyonensis 11RA and B. safensis 14A, which belonged to species not previously reported as 2,3-BD producers. Seventeen strains displayed cellulolytic activity, degrading carboxymethyl cellulose, HE-cellulose and beta-glucan, whereas 5 strains were also able to hydrolyze arabinoxylan, arabinan, galactomannan, xyloglucan, xylan and galactan (included in hemicellulose), as well as starch and fructans. The strain capacity to ferment lignocellulosic sugars to 2,3-BD corresponded to the activities of CAZymes engaged in the hydrolysis of the relevant polysaccharides. Regardless of species, Bacillus strains converted glucose, cellobiose, and mannose to 2,3-BD with higher concentration, productivity and yield than arabinose, xylose, and galactose. B. velezensis 5RB was the only strain, which was capable to produce 2,3-BD from all lignocellulosic sugars which corresponded to the presence of the highest extracellular endo-alpha-(1/5)-L-arabinanase, endo-1,4-beta-xylanase, 1,4-beta-xylosidase, and endo-beta-1,4-galactanase activity. The annotation of its genome showed the presence of 26 genes encoding glycoside-hydrolases allowing biomass degradation. In conclusion, the non-risk new isolated Bacillus strains are promising for 2,3-BD production from agro-industrial residues. When lignocellulose is used, the process would be more efficient if the substrate content is rich in cellulose and low in hemicelluloses. (C) 2020, The Society for Biotechnology, Japan. All rights reserved.