Rice-based co-culture systems offer sustainable agricultural benefits, yet stage-specific impacts on soil properties and grain quality remain underexplored. This study presented the first comprehensive assessment of the stage-specific effects under conventional tillage (CTL), rice-chicken (RC), rice-fish (RF), and rice-chicken-fish (RCF) systems on soil fertility, enzymatic activities, microbial communities, and grain quality. Our novel temporally explicit analysis revealed system- and stage-dependent modulation. RCF increased late-season organic matter by 10.4%, while RC consistently enhanced available potassium. Enzymatic activities exhibited distinct temporal shifts, with RF showing peak catalase activity at heading (0.47 mL g⁻¹ 30 min⁻¹), RC maintaining consistently higher invertase activity, and both RF and RCF displaying delayed urease peaks at filling (0.38 mg g⁻¹ 24 h⁻¹). Microbial communities were significantly restructured (ANOSIM, R² = 0.694, p < 0.001), with increased network complexity in co-cultures, particularly in RCF (95 nodes, 153 edges). Grain quality improvements included higher milling recovery (2.6–5.3%) in RC and elevated protein content (16.6%) in RF and RCF, along with reduced chalkiness (20–30%) across all co-cultures. Integrative analysis established linkages between soil properties (e.g., pH, organic matter, invertase), microbial taxa (e.g., Nitrospira, Syntrophus), and grain quality attributes. These findings provide mechanistic insights into soil-plant-microbe interactions and support the implementation of stage-specific management strategies for sustainable rice production systems.