Microcolony growth of Mycobacterium tuberculosis on agar proportion susceptibility testing is neither well-defined nor previously reported with fluoroquinolone susceptibility testing. We describe here M. tuberculosis microcolony growth with fluoroquinolones, and assess its clinical significance. We screened 797 M. tuberculosis isolates for ofloxacin resistance (2.0 μg/mL) by agar proportion; 19 ofloxacin-resistant and 38 ofloxacin-susceptible isolates were selected for more detailed susceptibility testing with ofloxacin, ciprofloxacin, levofloxacin (all at 2.0 μg/mL) and moxifloxacin (0.5 μg/mL). The 57 isolates were also tested at two concentrations both above and below the critical concentrations. Microcolonies were defined as colonies 0.2-0.4 mm in diameter; confirmed microcolonies were present on repeat testing. Of the 57 isolates tested in detail, 7 grew microcolonies, of which 2 (0.3% of all isolates tested) had confirmed microcolonies on repeat testing (6 tests performed, and microcolonies were present on at least 4). Both M. tuberculosis isolates were ofloxacin-resistant on screening, and had ofloxacin minimum inhibitory concentration (MIC) >8 μg/mL. The five other isolates were ofloxacin-susceptible on screening, but had regular colony growth (i.e., resistance) at the drug concentration that initially resulted in microcolonies (ofloxacin 0.5 or 1.0 μg/mL). Microcolonies were observed infrequently with fluoroquinolone susceptibility testing, but when confirmed, they were associated with drug resistance.