Virulent Mycobacterium kansasii (mainly subtype 1) may cause lung infections, whereas certain other strains (essentially subtype 3) are commonly non-pathogenic mycobacteria colonizing the human lower respiratory tract of patients. Determining the clinical significance of a strain isolated from a respiratory sample represents a major challenge for clinicians. Since some mycobacteria may use free-living amoebae as a training ground to select virulence traits, we wondered whether the Acanthamoeba castellanii amoeba could be used to determine the virulence of these intracellular bacteria. We investigated whether the growth and cytopathic effect of M. kansasii in A. castellanii correlate with the virulence of M. kansasii determined clinically and by subtyping. Pathogenic subtype 1 M. kansasii strains grew better in A. castellanii than non-pathogenic subtype 3 strains when considering both the number of bacteria per amoeba and the percentage of infected amoebae. Moreover, a subtype 3 M. kansasii strain isolated from blood culture, and thus considered pathogenic, was revealed to grow in A. castellanii similarly to pathogenic subtype 1 strains. These results suggest that amoebae may represent useful tools for testing the virulence of intracellular mycobacteria and other amoeba-resisting bacteria. This is important, since identification of novel bacterial virulence factors relies largely on in vitro assessment of virulence.