The long-term effect of organic and inorganic fertilizers on nutrient build-up and their relationship with microbial properties in a rice-wheat cropping sequence were studied in surface (0–15 cm) and subsurface (15–30 cm) soil samples. This experiment has been in progress since 1990 in the Department of Agronomy, CSK, Palampur with twelve treatments involving combinations of organic and inorganic fertilizers. In these treatment combinations, 25 and 50% of the recommended nitrogen levels were supplemented with organic sources, i.e. FYM (farm yard manure), green manure and wheat straw. The buildup of organic matter, and the total and available pools of nitrogen, phosphorus and sulphur were determined in surface (0–15 cm) and subsurface (15–30 cm) soil samples. The substitution of 25 and 50% nitrogen through organic fertilizers proved to be better than inorganic fertilizers alone. Amongst the organic sources, the substitution of FYM resulted in higher organic carbon, total and available nitrogen, phosphorus and sulphur than green manure or wheat straw. The substitution of 50% nitrogen through organic fertilizer was more effective than the substitution of 25% nitrogen. The relationships between the total and available pools of nutrients and the total microbial count, biomass carbon, microbial respiration, and the dehydrogenase and phosphatase enzymes were studied. The total pool of nutrients showed a high, positive, significant relationship with all these parameters.