THE MAIN concept of this study was to assess the potentiality of hydrolytic enzymes as virulence factors for Aspergillus species during aspergillosis infection process. Forty Aspergillus species were isolated from medical (15 isolates) as well as environmental isolates (25 isolates) from soils, outdoor and indoor environments. Extracellular proteases, phospholipases and esterase activities were measured. The pathogenicity of some Aspergillus species was in vivo assessed. It was represented in mean survival times, mortality percentages, fungal counts in different organs, and histopathological examination of lung tissue. The expression levels of protease, phospholipase and esterase genes were studied by real time PCR. Four physiologically significant isolates were selected out of forty and were identified up to molecular level. Aspergillus ochraceus fm90 recorded the highest pathogenicity as represented by mortality percentages and mean survival times of mice, while A. flavus fm90 was the least pathogenic one. Expression of genes of protease, phospholipase and esterase was found to be greater in A. ochraceus fm90 than in A. flavus fm90. It can be concluded that pathogenicity is probably related to physiological (enzymatic) activities of the isolates. Also, variation in expression levels of protease and phospholipase genes in A. ochraceus fm90 and A. flavus fm90 could denote their possible involvement in the pathogenicity process.