Antofloxacin (AFX) is a novel fluoroquinolone that has been approved in China for the treatment of infections caused by a variety of bacterial species. We investigated whether it could be repurposed for the treatment of tuberculosis by studying its in vitro activity. We determined the wild-type and non-wild-type MIC ranges for AFX as well as ofloxacin (OFX), levofloxacin (LFX), and moxifloxacin (MFX), using the microplate alamarBlue assay, of 126 clinical Mycobacterium tuberculosis strains from Beijing, China, of which 48 were OFX resistant on the basis of drug susceptibility testing on Löwenstein-Jensen medium. The MIC distributions were correlated with mutations in the quinolone resistance-determining regions of gyrA (Rv0006) and gyrB (Rv0005). Pharmacokinetic/pharmacodynamic (PK/PD) data for AFX were retrieved from the literature. AFX showed lower MIC levels than OFX but higher MIC levels than LFX and MFX on the basis of the tentative epidemiological cutoff values (ECOFFs) determined in this study. All strains with non-wild-type MICs for AFX harbored known resistance mutations that also resulted in non-wild-type MICs for LFX and MFX. Moreover, our data suggested that the current critical concentration of OFX for Löwenstein-Jensen medium that was recently revised by the World Health Organization might be too high, resulting in the misclassification of phenotypically non-wild-type strains with known resistance mutations as wild type. On the basis of our exploratory PK/PD calculations, the current dose of AFX is unlikely to be optimal for the treatment of tuberculosis, but higher doses could be effective.