Mesenchymal stem cells (MSC) and their niches, which are the factors secreted by MSC in culture in vitro, are considered to have a balancing and also therapeutic effect in numerous diseases. The treatment of tuberculosis (TB) caused by multidrug-resistant or extensively resistant Mycobacterium tuberculosis (MTB) strains is a challenging concern and may not be achieved by conventional treatment options. In the present study, we aimed to investigate the effect of MSC and their niches on MTB growth in culture medium. Frozen adipose-derived MSC were grown in culture medium until they became confluent. MSC and their niches, which were the factors secreted in two-day medium, were used as the conditioned medium and their effects on MTB were evaluated. MTB was maintained in liquid medium until a positive growth signal appeared on the Bactec MGIT 960 device (Becton Dickinson, Sparks, MD, USA). The growth periods (in days) were recorded. In the latter stage, the effects of 104 MSC in 100 µl medium, of the MSC niche that had been maintained with MSC in 100 µl medium for 48 h, and of their combined use on MTB growth were analyzed. After the appearance of a positive growth signal, the MTB growth and cell morphology in the tubes were examined under light microscopy. It was revealed that both MSC and MTB sustained viability in liquid medium. In the tubes administered with niches, the MTT assay showed MSC proliferation. However, in the tubes administered with a combination of MSC and niches or niches, MTB growth was inhibited and delayed. The retardant effect of MSC and their niches on MTB proliferation in liquid medium implicates that MSC and their niches can be used as therapeutic products in TB treatment. Substantiating the contribution of this excellent effect, which was found in in vitro studies, to TB treatment via 3D biomedical supports and animal model studies will have a strong effect for phase studies. Accordingly, we consider that the investigation of stem cell and its secretion, which has high reliability and low cost, in clinical studies is of paramount importance for ensuring patients’ quality of life.