To reduce transmission of tuberculosis (TB) in resource-limited countries where TB remains a major cause of mortality, novel diagnostic tools are urgently needed. We evaluated the fractional concentration of exhaled nitric oxide (FeNO) as an easily measured, noninvasive potential biomarker for diagnosis and monitoring of treatment response in participants with pulmonary TB including multidrug resistant–TB in Lima, Peru. In a longitudinal study however, we found no differences in baseline median FeNO levels between 38 TB participants and 93 age-matched controls (13 parts per billion [ppb] [interquartile range (IQR) = 8–26] versus 15 ppb [IQR = 12–24]), and there was no change over 60 days of treatment (15 ppb [IQR = 10–19] at day 60). Taking this and previous evidence together, we conclude FeNO is not of value in either the diagnosis of pulmonary TB or as a marker of treatment response.