The present study was conducted in a long-term field experiment (in progress since kharif 2009–10) at Department of Soil Science, Punjab Agricultural University, Ludhiana. The objective of the study was to investigate the impact of organic manure (biogas slurry) and fertilizer application on chemical fractions of zinc (Zn) and copper (Cu) under rice-wheat system. Treatments applied to rice-wheat system were arranged in a split-plot design with three replications. Main plot treatments applied to rice were four combinations of manure and fertilizers viz., nitrogen (N) @ 120 kg ha−1 without manure and phosphorus (P) fertilizer (M0N120P0), N @ 80 kg ha−1, P @ 30 kg ha−1 and manure @ 6 t ha−1 (M6N80P30), N @ 120 kg ha−1, P @ 30 kg ha−1 and no manure (M0N120P30) and N @ 80 kg ha−1, manure @ 6 t ha−1 without P-fertilizer (M6N80P0). Three sub-plot treatments applied in wheat included N @ 120 kg ha−1, K @ 30 kg ha−1 and three levels of Pfertilizer viz., P @ 30 kg ha−1 (P30), P @ 60 kg ha−1 (P60) and P @ 0 kg ha−1 (P0). Results revealed that the concentration of Zn and Cu was higher under the treatments where biogas slurry was applied along with fertilizers as compared to the sole application of fertilizers. The increase in water soluble and exchangeable (WSEX) fractions in organically treated plots indicated their mobilization from occluded fractions viz., crystalline iron oxide (CFeOX) and amorphous iron oxide (AFeOX) bound fractions. The residual (RES) fraction was the dominant portion of total Zn and Cu content of soil; whereas the contribution of WSEX fraction was limited. Organic matter bound fractions of Zn and Cu were the most important contributing towards their uptake by both rice and wheat crops.