Rice is a staple crop in Asia. In Cambodia, agroecological practices were introduced in Preah Vihear Province, testing organic systems with conventional tillage and no agrochemical inputs (CT) and cover cropping as green manure (GM) between rice cycles to enhance soil fertility and yield. This study compared the yield and quality of six Cambodian rice varieties (Phka Rumduol and the other five local cultivars) across 17 biplot systems (CT/GM). We assessed yield, physical quality (biomass, brokenness, moisture), nutritional quality (amylose and protein content) using NIR, and microbiological quality (seed-borne phytopathogens and mycotoxin-producing fungi) using PCR and amplicon sequencing. The results showed that GM practices significantly increased yield (12–19%) for all varieties except Phka Rumduol. Physical traits (biomass, brokenness, moisture) and amylose content of the nutritional traits differed between varieties but not between practices. Protein content varied both by varietal and practice effects: Neang Om had the highest protein (8.72 ± 0.6%), while CT seeds averaged 3% higher protein than GM. Diagnostic PCR detected common phytopathogens (C. oryzae, Pantoea spp., X. oryzae, B. glumae) and mycotoxin-producing fungi (Alternaria spp.), with differences across varieties but no consistent effect of practice. Amplicon sequencing of Phka Rumduol revealed additional phytopathogens and greater seed microbiome diversity in GM compared to CT. Overall, GM practices demonstrated strong potential to enhance yield and promote microbial diversity while maintaining seed quality consistent with market requirements. Meanwhile, CT systems preserved slightly higher protein levels. Long-term trials are needed before recommending wide-scale adoption to policymakers and farmers.