The goal of this work was to shed more light on a preliminary finding about the relationship between the substitution in the thioacyl part of thiobenzanilides and their antituberculous effect. Thus, we prepared a set of 14 derivatives, out of which eight had not yet been reported, and the compounds were evaluated for antimycobacterial activity on a panel of four Mycobacteria species, including Mycobacterium tuberculosis CNCTC My 331/88, Mycobacterium kansasii CNCTC My 235/80, Mycobacterium avium CNCTC My 330/88 and M. kansasii 6509/96. While the contribution of the substituents with differing electronic and lipophilicity characteristics in position 3 to the antituberculous activity was negligible, we found that unsubstituted position 4 in the thioacyl part appears to be a prerequisite for a thiobenzanilide derivative to possess appreciable biological activity.