This study examines humic substances in different land use systems across Rahuri, Pather, and Babulgaon soil series, investigating total acidity and acidic functional groups in humic acid (HA) and fulvic acid (FA). The highest total acidity (7.10 me g^-1), carboxylic group (4.40 me g^-1), and phenolic group (2.70 me g^-1) for HA were observed in Babulgaon series. Similarly, Fulvic acid in Babulgaon series exhibited the highest total acidity (9.25 me g^-1) and carboxylic group (6.45 me g^-1), while the phenolic group (3.95 me g^-1) peaked in Rahuri series. Surface soil in Babulgaon consistently displayed higher mean values for total acidity, carboxyl, and phenolic groups compared to Pather and Rahuri series. This trend persisted irrespective of soil series and land use, with these groups decreasing with increasing soil depth. Analyzing the impact of land use, guava orchard soils had the highest mean total acidity, carboxyl, and phenolic content for both HA and FA. Agriculture-cultivated lands followed, while fallow lands exhibited the lowest mean values. In conclusion, guava orchards exert a significant influence on soil properties, and specific numerical values highlight variations in humic substances across soil series and land use types.