In this work, a novel natural deep eutectic mixture was prepared from glucose, pregabalin and urea and used for the promotion of the synthesis of 1,8-dioxo-decahydroacridine and hexahydroquinoline derivatives through the one-pot Hantzsch condensation reaction. The new catalyst was characterized by several techniques including Fourier transform infrared spectroscopy (FT-IR), thermogravimetric analysis (TGA), derivative thermogravimetry (DTG), differential thermal analysis (DTA) and refractive index measurements (RI). Easy preparation of the reagent, green process, simple work-up procedure, high yields, and short reaction times are among the most important advantages of the reported protocol. In this research Antibacterial activities of the prepared deep eutectic mixture and new synthesized Hantzsch derivatives were also studied against the Bacillus subtilis (Gram-positive) and Escherichia coli (Gramnegative) by the agar well diffusion method.