A sonotrode ultrasound-assisted extraction of phenolic compounds from olive leaves has been developed using a Box–Behnken design to optimize the effects of solvent composition and ultrasound parameters. The determination of single phenolic compounds was performed by HPLC–MS and the highest recovery in total compounds, oleuropein and hydroxytyrosol was achieved using EtOH/H2O (55:45, v/v), 8 min and 100% of amplitude. The optimal conditions were applied on leaves from seven olive cultivars grown under the same conditions and the results were compared with those found by using a conventional ultrasonic bath, obtaining no statistical differences. Moreover, antioxidant activity by FRAP, DPPH and ABTS in these olive leaf extracts was evaluated and they exhibited a significant correlation with oleuropein and total phenolic content. All cultivars of olive leaf extracts were found to be active against S. aureus and methicillin-resistant S. aureus with minimum bactericidal concentration (MBC) values) that ranged from 5.5 to 22.5 mg mL−1. No extracts showed antimicrobial activity against C. albicans. The percentages of mycelium reduction in B. cinerea ranged from 2.2 and 18.1%. Therefore, sonotrode could be considered as an efficient and fast extraction technique that could be easily scaled-up at industrial level, thus allowing for olive leaves to be revalorized.