The current study evaluated the effect of powder fractionation based on particle size on the chemical composition of macronutrients such as proteins and sugars, on the phytochemical properties (total content of polyphenolic compounds, vitamin C, and antioxidant activity), on preservation capacity (water activity), powder functional properties (water absorption capacity and water solubility index), and physicochemical properties (particle size distribution and moisture content) of blackthorn berry (Prunus spinosa) powders. The fruits were separated from the plant material and seeds, dried, and then ground using an universal mill for dry materials. Eight fractions were obtained after sieving on sieves with different mesh sizes, such as 1 mm, 0.8 mm, 0.630 mm, 0.450 mm, 0.315 mm, 0.200 mm, and 0.125 mm. The grinding/sieving procedure was effective in separating Prunus spinosa powder into sufficiently different size classes. The maximal moisture content and water activity were 5.61% and 0.250, respectively, showed good preservation from a microbiological point of view, and ensured the prevention of oxidation of biologically active compounds of blackthorn berry powders. For samples with reduced particle sizes, the powder functional properties were greatly improved. The total phenolic content, carbohydrates, and antioxidant activity showed significantly different values for some particle size classes compared to the un-sieved sample. A considerable content of vitamin C was presented in the fraction with large particle sizes, precisely because they did not undergo intense degradation processes. Therefore, the technique of grinding and successive sieving proved effective in enhancing the physicochemical and functional characteristics of powdered blackthorn berries, particularly for smaller particles.