The current study investigated whether ABO blood mistyping of human biological samples is induced by soil bacteria. A total of 380 bacterial strains were isolated from 50 discrete soil samples using human blood agar, and glycosidase activity evaluated for all strains using 4-nitropheny glycosides (4-nitrophenyl n-acetyl-α-D-galactosaminide, 4-nitrophenyl-α-D-galactopyranoside, 4-nitrophenyl-α-L-fucopyranoside) as substrates. Thirteen strains possessed α-galactosidase activity, and 16S rRNA sequence analysis revealed a close relatedness to the genus Bacillus. An indirect competitive enzyme-linked immunosorbent assay confirmed seven strains exhibited type B antigen degradation activity. These results demonstrated that 1.8% of the bacteria isolated from soil, were Bacillus sp., possessed galactosidase activity, and had the potential to cause ABO blood mistyping.