Objective: To determine at two distinct time points the prevalence of resistance to ofloxacin (OFX), the representative class drug of fluoroquinolones (FQs), in M. tuberculosis isolates susceptible to first-line drugs. Results: There were 279 M. tuberculosis isolates from the two cohorts (2004–2005: 238 isolates; 2017: 41 isolates) that underwent OFX drug-susceptibility testing (critical concentration: 2 µg/ml). Of 238 isolates in Cohort 1, no resistance to OFX was detected (95% CI 0–0.016); likewise, in Cohort 2, no resistance to OFX was detected in 41 isolates (95% CI 0–0.086). Our findings suggest that FQ use remains a viable option for the treatment of first-line drug-susceptible TB in Peru.