Cryptococcus neoformans species complex is an opportunistic yeast that causes life-threatening infections in immunocompromised hosts. The aim of the study was to investigate the epidemiological distribution, antifungal susceptibility and molecular characterization of C. neoformans species complex Croatia and Kosovo. During 2013-2016, 709 samples were collected from trunk hollows and birds’ excreta in the soil beneath the trees in different geographical locations. Four C. neoformans species complex isolates was found in environmental samples of tree hollows collected in two cities, Krk and Rijeka, on Croatian coast, but not in Kosovo. The tested antifungal compounds demonstrated high in vitro activity against environmental C. neoformans species complex isolates. Three C. gattii species complex were isolated in both, Croatia and Kosovo and belonged to wild type for all tested antifungals as well according to ECVs. Three out of four (3/4; 75%) of C. neoformans species complex isolates in Croatia belonged to serotype A and all (4/4, 100%) had mating type α. The four C. neoformans species complex isolates from Croatia were classified into two different molecular types, VNI (n = 3) and VNIV (n = 1). This is the first report proving the presence of C. neoformans species complex in the environment of Croatia and determining their susceptibility to antifungals. The results of the study show the potential risk of exposure for inhabitants, especially on the Croatian coast, to molecular types VNI and VNIV, which can be expected in clinical cases of cryptococcosis. Although a limitation of this study is the small number of detected environmental isolates antifungal susceptibility pattern of C. neoformans species complex suggests that no strains with low susceptibility to the most common antifungals should be expected in patients with cryptococcosis.