Plant-growth-promoting bacteria (PGPB) and biochar have attracted increasing attention for remediating the combined pollution of arsenic (As) and cadmium (Cd) and promoting selenium (Se) biofortification. However, their differing effects on the bioavailability of As, Cd, and Se and their absorption by rice are still poorly understood. In this study, PGP Agrobacterium sp. T3F4 with Se- oxidizing capacity and corn straw biochar were applied to natively polluted paddy soil. Strain T3F4 reduced the bioavailability of As in soil but increased bioavailable Se, decreasing the As content in rice by 16.8% and improving the Se content of rice by 54.5% (p <
.05). Application of 2.5% biochar stimulated iron (Fe) plaque formation of the root and immobilized As and Cd in the soil, decreasing the As and Cd absorption of rice by 14.7% and 40.3%, respectively (p <
.05). Application of 5.0% biochar achieved similar mitigation effects for As and Cd but also decreased the Se content in rice by 60.6% by reducing bioavailability. This decrease in Se uptake was mitigated when 5.0% biochar was co-applied with strain T3F4. Notably, applying strain T3F4 also alleviated the oxidative stress on rice plants and enhanced soil enzyme activities, contributing to a substantial increase in grain yield in the polluted paddy soil. The adverse effects of 5.0% biochar on soil health and grain yield were mitigated by the co-application of strain T3F4. Our results provide new insights into applying PGPB and biochar for Se biofortification and As and Cd remediation in paddy soil.