The purpose of this study was to isolate and identify the most important species belonging to Aspergillus genus that cause aspergillosis disease in Riyadh city, Saudi Arabia and determine the best specimens taken from patients to isolate the Aspergillus, as well as to specify the best antifungal drugs for its inhibition, also to identify which of these isolate species are more resistant to these antifungal agents. A total of 53 samples was collected from patients initially diagnosed as infected with Aspergillosis disease. These samples were from sputum, tracheal aspirate, nasopharyngeal aspirate, bronchoaveolar lavage and lung tissue. Direct examination, culturing process, isolation of the fungus were performed to identify the isolates by their micro- and marocharacteristics. To confirm the identification, the isolates were sent to Assiut University Mycological Center (AUMC) in Egypt. In vitro susceptibility test of different antifungal (Amphotericin B, Anidulafungin, Caspofungin, Fluconazole, 5-Flucyosine, Itraconazole, Micafungin, Posaconazole and Voriconazole) for isolated Aspergillus was carried out. The results revealed that the most fungal isolates were obtained from sputum samples followed by aspirate samples from trachea. The cause of aspergillosis disease was confined to the five following species: A. fumigatus, A. flavus, A nidulans, A. niger and A. terreus. The most frequent isolate was A. fumigatus. The antifungal caspofungin and anidulafungin were the most effective ones, whereas fluconazole and 5-flucyostine were the least effective antifungal drugs. It was also found that A. fumigatus was the most resistant species among all studied isolates.