In this study, N-[3-(3-amino-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-4-R-thiazol-2(3H)-ylidene]-2,6-difluorobenzamide derivatives as new 1,4-naphthoquinone thiazole hybrids were synthesized by reacting of N-[(3-amino-1,4-dioxo-1,4-dihydronaphthalen-2-yl)carbamothioyl]-2,6-difluorobenzamide with various α-bromoketones in 76–92% yields. Their molecular structures were characterized by 1H NMR, 13C NMR, 19F NMR, FT-IR, and HRMS, and the stereochemistry of one of the hybrids was determined by single crystal x-ray diffraction study. These synthesized new compounds (3a–e) were found to be effective inhibitor molecules for cholinesterases (butyrylcholinesterase (BChE) and acetylcholinesterase (AChE)), and carbonic anhydrase I and II (hCA I and hCA II) enzymes. KI values were found to be in the range of 45.03–84.43 nM for BChE, 26.12–98.42 nM for AChE, 67.86–161.60 nM for hCA I, and 55.27–87.48 nM for hCA II. The acid dissociation constants (pKa) of 1,4-naphthoquinone thiazole hybrids were determined in 25% (v/v) DMSO:water (25.0 ± 0.1 °C, I = 0.1 M by NaCl). Three pKa values for each hybrid were calculated with the HYPERQUAD program from the data obtained as a result of potentiometric titrations. The results obtained from molecular docking studies indicate that the compounds in question favorably fit within the active sites of hCAs and ChEs. Additionally, the acceptability of these compounds, as determined by Lipinski's and Jorgensen's rules, was estimated using the ADME/T results. Based on these estimations, it can be concluded that the synthesized molecules have the potential to be developed as effective and safe inhibitors of hCAs and ChEs, thus making them suitable lead agents for glaucoma and Alzheimer's disease.