The cascade dehydration of glucose to 5-hydroxymethylfurfural (HMF) was carried out in water over a series of Nb₂O₅ catalysts, which were derived from the thermal treatment of niobic acid at 300 and 550 °C, under air or inert atmosphere. Amorphous niobic acid showed high surface area (366 m²/g) and large acidity (2.35 mmol/g). With increasing the temperature of the thermal treatment up to 550 °C, the amorphous Nb₂O₅ was gradually transformed into a pseudohexagonal phase, resulting in a decrease in surface area (27–39 m²/g) and total acidity (0.05–0.19 mmol/g). The catalysts’ performance in cascade dehydration of glucose realized in pure water was strongly influenced by the total acidity of these materials. A remarkable yield of 37% HMF in one-pot reaction in water was achieved using mesoporous amorphous niobium oxide prepared by thermal treatment of niobic acid at 300 °C in air. The best-performing catalyst displayed a total acidity lower than niobic acid (1.69 mmol/g) which afforded a correct balance between a high glucose conversion and limited further conversion of the target product to numerous polymers and humins. On the other hand, the treatment of niobic acid at 550 °C, independently of the atmosphere used during the sample preparation (i.e., air or N₂), resulted in Nb₂O₅ catalysts with a high ratio of Lewis to Brønsted acid sites and poor total acidity. These materials excelled at catalyzing the isomerization step in the tandem process.