Fluoroquinolones (FQs) manifest bactericidal activity in a concentration-dependent manner as they inhibit DNA gyrase and topoisomerase activity that are crucial in the replication process. They are the key component of a multidrug-resistant tuberculosis treatment regimen. FQs are effective against bacterial infections that cause skin, urinary tract, and prostate-related infections. Ofloxacin is also being tried as part of modified Multidrug Treatment (MDT) regimens against leprosy. This study examined the accumulation of ofloxacin in M. smegmatis, M. kansasii, and M. tuberculosis. These mycobacteria were grown in Sauton’s liquid medium up to the exponential phase. Finally, cells were harvested, cleaned by washing, and resuspended. Suspensions were incubated with ofloxacin at 37 °C, and ofloxacin concentration in supernatants was observed using spectrofluorimetry. Ofloxacin concentrations were also observed in the absence or presence of Carbonyl cyanide m-chlorophenylhydrazone (CCCP), which is a proton motive force inhibitor. Steady state concentration (SSC) of ofloxacin was achieved in M. smegmatis (03 minutes), M. kansasii (03 minutes), and M. tuberculosis (05 minutes), and the ofloxacin accumulation level was 102 ng/mg, 100 ng/mg, and 107 ng/mg (dry weight of the bacilli), respectively. Ofloxacin accumulation was found at 10 µg/ml concentrations; however, CCCP addition was unable to influence the ofloxacin accumulation. This study concludes that ofloxacin accumulation is a simple diffusion in M. smegmatis, M. kansasii, and M. tuberculosis. Through simple diffusion, ofloxacin is transported across the mycobacterial cell envelope, inhibiting type II DNA isomerase (gyrase), which is required for DNA replication. Thus, it is effective in the treatment of disease.