Background Multidrug-resistant infections due to Mycobacterium abscessus often require complex and prolonged regimens for treatment. Here, we report the evaluation of a new ex vivo antimicrobial susceptibility testing model using organotypic cultures of murine precision-cut lung slices, an experimental model in which metabolic activity, and all the usual cell types of the organ are found while the tissue architecture and the interactions between the different cells are maintained. Methods Precision cut lung slices (PCLS) were prepared from the lungs of wild type BALB/c mice using the Krumdieck® tissue slicer. Lung tissue slices were ex vivo infected with the virulent M. abscessus strain L948. Then, we tested the antimicrobial activity of two drugs: imipenem (4, 16 and 64 μg/mL) and tigecycline (0.25, 1 and 4 μg/mL), at 12, 24 and 48 h. Afterwards, CFUs were determined plating on blood agar to measure the surviving intracellular bacteria. The viability of PCLS was assessed by Alamar Blue assay and corroborated using histopathological analysis. Results PCLS were successfully infected with a virulent strain of M. abscessus as demonstrated by CFUs and detailed histopathological analysis. The time-course infection, including tissue damage, parallels in vivo findings reported in genetically modified murine models for M. abscessus infection. Tigecycline showed a bactericidal effect at 48 h that achieved a reduction of > 4log10 CFU/mL against the intracellular mycobacteria, while imipenem showed a bacteriostatic effect. Conclusions The use of this new organotypic ex vivo model provides the opportunity to test new drugs against M. abscessus, decreasing the use of costly and tedious animal models.