A series of quinolones with substitutions at the 8 position has been prepared as part of a study to examine the relationship between structural modifications at this position and activity against mycobacteria. The compounds were prepared by procedures described in the literature and were evaluated for their activities against Mycobacterium fortuitum and Mycobacterium smegmatis. The activities of the compounds against these two organisms were used as a measure of Mycobacterium tuberculosis activity. The results demonstrate that the contribution of the 8 position to antimycobacterial activity was dependent on the substituent at N-1 and was in the order (i) COMe ≃ CBr > CCl > CH ≃ CF ≃ COEt > N > CCF3 when N-1 was cyclopropyl; (ii) N ≃ CH > CF > COMe when N-1 was 2,4-difluorophenyl; (iii) N ≤ CH when N-1 was tert-butyl; and (iv) N > CH when N-1 was ethyl. In general, derivatives with piperazine substitutions at C-7 were slightly less active against mycobacteria than the analogs with pyrrolidine substitutions, regardless of the pattern of substitution at the 8 position. Several of the best compounds were evaluated for their potential side effects as well as their activities against Mycobacterium aurum, Mycobacterium avium-M. intracellulare, and M. tuberculosis. These agents exhibited biological profiles similar to or better than those of the positive controls ciprofloxacin and sparfloxacin.