The chemical composition of the essential oil of the Sardinian Rosmarinus officinalis L. obtained by hydro distillation and steam\hydro distillation was studied using GC-FID and MS. Samples were collected at different latitude and longitude of Sardinia (Italy). The yields ranged between 1.75 and 0.48% (v/w, volume/dry-weight). A total of 30 components were identified. The major compounds in the essential oil were alpha-pinene, borneol, (-) camphene, camphor, verbenone, and bornyl-acetate. Multivariate analysis carried out on chemical molecular markers, with the appraisal of chemical, pedological, and random amplified polymorphic DNA data, allows four different clusters to be distinguished. The antimicrobial and antifungal tests showed a weak activity of Sardinian rosemary. On the other hand, an inductive effect on fungal growth, especially toward Fusarium graminearum was observed.