Background Data correlating in vitro drug susceptibility of Microsporum canis with clinical outcomes of its infections are lacking as well as the most suitable inoculum and incubation time in broth microdilution assays. Objectives and Methods M. canis strains were collected from animal hosts that tested positive (Group I; n=13) and negative (Group II; n=14) to this pathogen following itraconazole (ITC) therapy. In vitro ITC susceptibility was assessed according to the Clinical Laboratory Standards Institute (CLSI M38-A2) methodology using conidia, hypha-conidia and arthroconidia at 3 and 7 days of incubation in order to assess the most suitable inoculum and incubation time. Successively, ketoconazole (KTC), voriconazole (VRC) terbinafine (TRB), posaconazole (PSZ), fluconazole (FLC) and griseofulvin (GRI) susceptibilities were assessed using the chosen inoculum. Results The MIC values of ITC after three day-incubation were equal than those recorded after seven day-incubation. ITC MICs were ≤1μg/ml for strains from Group II and >1μg/ml for those of Group II only when conidia were used. All strains showed high susceptibility to VRC, POS, TEB and low susceptibility to ITC, KTC, GRI and FLC regardless of the source and incubation time. Conclusions and clinical importance Results suggest that correlation between the in vitro results and clinical outcome was observed only by incubating conidia for 3 days at 30±2oC.These conditions might be most suitable to assess in vitro susceptibility of M. canis and assist in determining the occurrence of drug resistance and cross -resistance phenomena.