The potential relationship between the presence of gliotoxinogenic Aspergillus species and the surrounding environment of lactating cows. Aspergillus fumigatus is the main etiologic agent causing invasive aspergillosis. Also, the A. fumigatus complex may be a cause of invasive aspergillosis. In this study, A.fumigatus strains were isolated from lactating cow’s milk in Taif, Saudi Arabia, and identified using morphological criteria via cultivating Aspergillus species inoculations on Czapek agar, and malt extract agar (MEA) plates, followed by examining the activity of the Rosmarinic acid against Aspergillus species using diffusion disk and MIC assays. Also interesting was the molecular docking of Rosmarinic acid against 14- alpha sterol demethylase Cyp51B enzyme which considers a target site for fungal infection. The result has shown that morphologic identification of the strains isolated from lactate cow milk displayed two classes of Aspergillus species: Aspergillus fumigatus which has a colony blue-green in front and converts into colorless in reverse with a diameter is 2-3 cm and a Vesicle diameter 23 μm, the second class was Aspergillus brevipes which has a colony color in front is yellow and convert into brown in reverse with diameter is 2 cm and Vesicle diameter 14 μm. The inhibition zone of A. fumigatus and A. brevipes were 17 and 15 nm respectively after the strains were investigated with RA. The MIC value of RA against Aspergillus fumigatus and Aspergillus brevipes isolated from cow milk was 321 and 326 mg, respectively. The 14-alpha sterol demethylase Cyp51B enzyme was inhibited by RA via the formation of two hydrogen bonds with Ser511 and Arg510 respectively.