•Lactic acid-based eutectic mixtures were studied for hop’s active compounds recovery.•Structural analysis was performed by FTIR and Raman spectroscopic techniques.•Eutectic mixtures improved the extraction efficiency of hop’s bioactive compounds.•The highest antimicrobial activity was recorded against P. aeruginosa. In the present study, natural deep eutectic solvents were used to increase the extraction efficiency (EE) of bioactive compounds from hop (Humulus lupulus L) cones under ultrasounds irradiation. Three lactic acid-based deep eutectic solvents (namely LaDES) were developed, physicochemical characterized and their EE values compared with those of a control extract, obtained using aqueous methanol (80%) acidified with HCl (0.1%). The supramolecular organization of LaDES and their extracts were analysed using FTIR spectroscopy and Raman spectroscopy to clarify the interactions among LaDES components and between LaDES and the bioactive components of the plant matrix. The phytochemical profiles of green extracts were evaluated using both spectrophotometric assays and HPLC-DAD analysis and the results were compared with those from the control samples. Among the different combinations of LaDES tested, lactic acid: sucrose showed the highest EE against hop bitter acids (α-acids: 8.9 ± 0.1% w/w; β-acids: 3.2 ± 0.3% w/w), while lactic acid: urea extracted more non-phenolic pigments (total chlorophyll: 8.79 ± 0.09 mg g−1 d.b.; total carotenoids: 2.22 ± 0.02 mg g−1 d.b.) than the other two LaDES combinations and control. Polyphenols were better extracted by lactic acid: glycine than the other solvents tested (71.2 ± 0.1 mg gallic acid equivalents g−1 d.b.), also showing the highest antiradical potential against both ABTS+ and DPPH (EC50_ABTS: 0.63 ± 0.01 μg; EC50_DPPH: 69 ± 2 μg). Finally, all LaDES inhibited both Gram- positive and negative bacteria, while non activity was recorded against tested yeasts.