This study aimed to evaluate the dynamics of culture filtrate dependent subpopulations of Mycobacterium tuberculosis in a prospective cohort study following 17 patients through a standard 6-month anti-tuberculosis regimen, performing monthly sputum collection. We performed the limiting dilution method with culture filtrate supplementation of liquid media in pre- and post-treatment sputum samples to assess the bacillary load and to evaluate the Mycobacterium tuberculosis subpopulation dynamics within the 6-months standard anti-tuberculosis regimen. We found that supplementation increased the bacillary load by 30% in pre-treatment samples (p = 0.0005) and 35% in samples after one month of treatment (p = 0.0977). We found a weak linear correlation between the decrease of Mycobacterium tuberculosis growth in liquid media with and without culture filtrate supplementation (ρ = 0.54; p = 0.026). None of the patients had bacilli recovery after two months of treatment. Our study constitutes the first follow-up regarding Mycobacterium tuberculosis subpopulation dynamics throughout a standard 6-month anti-tuberculosis treatment and also supports the use of culture filtrate to increase bacillary load in liquid media. Moreover, it highlights that any new treatment regimens should test the efficacy of the drugs in all Mycobacterium tuberculosis subpopulations.