To reveal rare phenotypes in bacterial populations, conventional microbiology tools should be advanced to generate rapid, quantitative, accurate, and high-throughput data. The main drawbacks of widely used traditional methods for antibiotic studies include low sampling rate and averaging data for population measurements. To overcome these limitations, microfluidic-microscopy systems have great promise to produce quantitative single-cell data with high sampling rates. Using Mycobacterium smegmatis cells, we applied both conventional assays and a microfluidic-microscopy method to reveal the antibiotic tolerance mechanisms of wild-type and msm2570::Tn mutant cells. Our results revealed that the enhanced antibiotic tolerance mechanism of the msm2570::Tn mutant was due to the low number of lysed cells during the antibiotic exposure compared to wild-type cells. This is the first study to characterize the antibiotic tolerance phenotype of the msm2570::Tn mutant, which has a transposon insertion in the msm2570 gene-encoding a putative xanthine/uracil permease, which functions in the uptake of nitrogen compounds during nitrogen limitation. The experimental results indicate that the msm2570::Tn mutant can be further interrogated to reveal antibiotic killing mechanisms, in particular, antibiotics that target cell wall integrity.