A collection of 48 clinical Cryptococcus neoformans isolates from Croatia was investigated retrospectively using in vitro antifungal susceptibility testing and molecular biological techniques to determine mating type and serotype by PCR and amplified fragment length polymorphism (AFLP) genotyping. These isolates were obtained from 15 patients: ten were human immunodeficiency virus (HIV)-negative (66.7 %) and five were HIV-positive (33.3 %). From five patients, only one isolate was available, whilst from the other ten patients, two to 11 isolates were isolated sequentially. Antifungal susceptibility was tested by a broth microdilution method. Serotype A (genotype AFLP1) and serotype D (genotype AFLP2) were both found in six patients (40 % each), and serotype AD (genotype AFLP3) in three (20.0 %) patients. Mating type α (n = 12; 80.0 %) predominated and α/a hybrids were identified in 20.0 % of patients diagnosed with cryptococcosis. Two AFLP genotypes of C. neoformans were isolated during a single episode from one patient. The in vitro antifungal MIC(90) and susceptibility ranges for C. neoformans isolates were 0.5 µg ml(-1) (range 0.031-0.5 µg ml(-1)) for amphotericin B, 4 µg ml(-1) (range 1-4 µg ml(-1)) for flucytosine and fluconazole, 0.25 µg ml(-1) (range 0.031-0.5 µg ml(-1)) for itraconazole and 0.062 µg ml(-1) (range 0.031-0.25 µg ml(-1)) for voriconazole.