Cryptococcus neoformans and Cryptococcus gattii are pathogenic yeasts that cause cryptococcosis. These fungi were commonly associated with pigeon droppings and plant materials. The habitat of these pathogens has not been yet studied in Tunisia, although the ecology of these yeasts must be elucidated in order to establish surveillance programs and to prevent infections. The aim of this survey was to recover C. neoformans and C. gattii environmental isolates from pigeon droppings and plant materials in different areas of Sfax region, Tunisia. Nine hundred and fifty samples from leaves, wood, flowers, fruits and soil around trunk bases of 40 almond (Prunus dulcis) and 60 eucalyptus trees were collected as well as 250 pigeon droppings samples from different sites: buildings (n = 150), houses (n = 50) and zoo (n = 50). The identification of Cryptococcus neoformans complex was confirmed using the ID32C auxanogram panel (BioMérieux, Marcy l'Etoile, France); species were determined by multiplex PCR using the CN70 and CN49 primers, and mating type was determined by PCR. C. neoformans was recovered from 26 specimens of pigeon droppings (10.4%). This yeast was obtained more frequently from dry droppings (9.2%) than from moist droppings (1.2%). The mating type was determined. All the 31 environmental strains of C. neoformans and C. gattii were MATα. Out of 700 samples tested from 100 trees, only 5 isolates of Cryptococcus neoformans species complex were recovered (0.6%), two isolates of C. gattii and one isolate of C. neoformans were recovered from the wood of E. camaldulensis trees, and only two isolates of C. gattii were recovered from the wood of almond trees (Prunus dulcis Mill. var. zaaf and var. achek). These two Tunisian almond tree varieties were recorded for the first time in Africa as hosts for C. gattii. These results add new information to the ecology and epidemiology of C. neoformans species complex in Tunisia.