Human pathogens belonging to the Nakaseomyces clade include Candida glabrata sensu stricto, Candida nivariensis and Candida bracarensis. Their highly similar phenotypic characteristics often lead to misidentification by conventional laboratory methods. Therefore, limited information on the true epidemiology of the Candida glabrata species complex is available. Due to life-threatening infections caused by these species, it is crucial to supplement this knowledge. The aim of the study was to estimate the prevalence of C. bracarensis and C. nivariensis in a culture collection of C. glabrata complex isolates. The study covered 353 isolates identified by biochemical methods as C. glabrata, collected from paediatric and adult patients hospitalised at four medical centres in Southern Poland. The multiplex PCR was used to identify the strains. Further species confirmation was performed via sequencing and matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) analysis. One isolate was recognised as C. bracarensis (0.28%). To our knowledge, it is the first isolate in Poland. C. glabrata sensu stricto species has been confirmed for all the remaining isolates. No C. nivariensis was found. Our study has shown that the prevalence of C. nivariensis and C. bracarensis strains is infrequent. However, it should be emphasised that the incidence of these strains may differ locally and depend on environmental factors and the population.