BshB, a key enzyme in bacillithiol biosynthesis, hydrolyses the acetyl group from N-acetyl-glucosamine-malate to generate glucosamine-malate. In Bacillus anthracis, BA1557 has been identified as the N-acetyl-glucosamine-malate deacetylase (BshB), however high content of bacillithiol (~70%) was still observed in Bacillus anthracis ?BA1557 strain. Genomic analysis led the proposal that another deacetylase could exhibit cross functionality in bacillithiol biosynthesis. Herein, BA1557, its paralog BA3888 and orthologous B. cereus enzymes, BC1534 and BC3461, have been characterized for their deacetylase activity towards N-acetyl-glucosamine-malate, thus providing biochemical evidence for this proposal. In addition, the involvement of deacetylase enzymes is also expected in bacillithiol-detoxifying pathways through formation of S-mercapturic adducts. The kinetic analysis of bacillithiol-S-bimane conjugate favors the involvement of BA3888 as the B. anthracis bacillithiol-S-conjugate amidase (Bca). The high degree of specificity of this group of enzymes for its physiological substrate, along with their similar pH vs activity profile and Zn2+-dependent catalytic acid–base reaction provides further evidence for their cross-functionalities.