Knowledge of long-term phosphorus behavior is essential to improve soil structure, nutrient supply potential, and the sustainability of cropping systems. A 45-year long-term experimental trial was used to observe organic phosphorus fractionation and its effects on soil aggregation and nutrient distribution at three depths (0–20, 20–40, and 40–60 cm) in Vojvodina Province, Serbia, under maize monoculture and maize/barley rotation. Five fertilizing systems were studied, including Control, NPK, NPK + maize remains, NPK + manure, and NPK + manure in rotation. Soil aggregates were fractionated into four size categories (>2000, 2000–250, 250–53, and <53 μm) using a wet sieving method. The samples were analyzed for main indicators, including different forms of phosphorus, total and available (PT and PA), as well as its organic forms (Labile Po, Biomass Po, Mod. Labile Po, Fulvic acid Po, Humic acid Po, and Resistant Po), and other fertility parameters. Significant differences in total and available phosphorus as well as all observed organic phosphorus fractions were evident between treatments with and without organic amendments, particularly in the 0–20 and 20–40 cm soil layers. Moderately labile P forms were dominant across all treatments, while labile forms constituted a smaller proportion. The most notable differences between treatments were observed in the labile and moderately labile forms, as well as in the resistant form of organic phosphorus. Manure application led to increased nutrient content in macroaggregates (>250 μm) compared to microaggregates. Microaggregates (<250 μm) were predominant across all depths, while stable structural aggregates did not show a significant increase after manure application. PCA highlighted significant correlations between soil characteristics, including total and available P, total organic carbon, clay content, and enzyme activity, across different aggregate sizes and organic P fractions. Overall, long-term mineral fertilization combined with organic amendment application induced variations in phosphorus fractions and the content of carbon, nitrogen, and phosphorus associated with aggregates in the first two soil layers, except for aggregate size classes.