Olive (Olea europaea L.) leaves are an abundant byproduct in the olive oil industry characterized by high quantities of phenols. The content of these molecules in olive leaves may vary according to several factors, including cultivar, olive tree alternate bearing cycle, and seasonal variations. In the present study, leaves belonging to five Italian cultivars (‘Biancolilla’, ‘Nocellara Etnea’, ‘Nocellara Messinese’, ‘Nocellara Siracusana’, ‘Zaituna’), cultivated in the same area, were collected from January to August and individually subjected to an appropriate extraction assay. Each extract was analyzed by means of HPLC/DAD/ESI-MS to determine its phytochemical profile and assess inter cultivar and seasonal variations. Moreover, total phenolics, ORAC, and DPPH assays were performed, in order to evaluate the Total Polyphenol Content and antioxidant potential. Seventeen different metabolites belonging to the subclasses of simple phenols, secoiridoids, hydroxycinnamic acids, and flavonoids were identified, quantified, and used as markers to monitor profile variations. The data obtained have shown that secoiridoids, particularly oleuropein, can be regarded as key molecules to discriminate among different cultivars and to assess variability in olive leaves during the flower and fruit development stage. All of the samples evidenced a remarkable antioxidant activity in terms of Radical Scavenging Activity, and as expected, some differences were observed among cultivars and depending on the harvest period through ORAC assay.