Although there has been an increase in the cultivation of highbush blueberry (Vaccinium corymbosum L.) worldwide for several years now, the availability of suitable soils for this species remains a problem. Highbush blueberry is a plant that requires acidic soils (pH 3.8–5.5), which are well aerated and have a stable level of groundwater and high humus content. In the present study, substances such as urea phosphate fertilizer, sulfur, sulfuric acid, and phosphogypsum were used to acidify three soils: peat, loamy sand, and loamy silt. The study aimed to lower the pH of the tested soils and optimize this parameter to cultivate highbush blueberry. The resulting changes in pH, content of macro- and micro-elements, and enzymatic activity were evaluated. Acidifying substances mitigated peat and loamy sand’s reaction to highbush blueberry requirements, while the reaction of loamy silt was changed only slightly, which made this soil unsuitable for plant cultivation. Sulfur dust acidified the examined soils rapidly and to the highest degree, followed by urea phosphate and phosphogypsum, while the weakest acidification was achieved with sulfuric acid solutions. The salt concentration of the soil was increased the most by the highest dose of phosphogypsum, which indicated that it could not be used to acidify soil for the cultivation of highbush blueberry. Among the acidifying substances, only urea phosphate showed a stimulating effect on the soils’ enzymatic activity, whereas others did not significantly affect or decrease this parameter.