Trimetallic NiMoW/Al2O3 catalysts based on mixed H4[SiMonWn-12O40] (n = 1, 3, 6, and 9) Keggin-type heteropolyacids (HPAs) were synthesized by incipient wetness impregnation of alumina with aqueous solutions of mixed HPAs. For comparison purposes, trimetallic samples were prepared from a mixture of monometallic H4[SiMo12O40] and H4[SiW12O40] HPAs with a Mo/W ratio corresponding to the mixed MoW HPAs. The catalysts were sulfided by a liquid phase method and tested in the model reactions of dibenzothiophene hydrodesulfurization and naphthalene hydrogenation, with subsequent addition of quinoline to study the effect of inhibition of target reactions. Further, the catalysts were tested in the hydrotreating of straight-run gas oil to evaluate the efficiency of catalysts on real feedstocks. In order to link catalytic performances with the preparation method and Mo/W ratio, the catalysts were fully characterized by high-resolution transmission electron microscopy, X-ray photoelectron spectroscopy, high-angle annular dark-field imaging, and quick X-ray absorption spectroscopy. It was found that the Mo/W atomic ratio of the structure-forming metals in the active phase, deriving from the Mo/W ratio in the HPA precursor, directly affects the ratio of the hydrogenation and hydrodesulfurization performances. For feedstock with a high concentration of N-containing compounds, it is necessary to use mixed NiMoW systems with a high tungsten percentage to reduce the inhibitory effect and ensure the quality of the products. The use of new mixed MoW Keggin HPAs has made it possible to enhance the synergistic effect in trimetallic NiMoW catalysts due to the closer interaction between Mo and W, which increased the sulfidation degree of metals and also contributed to the formation of highly active mixed NiMoWS sites.