Agronomic selenium (Se) biofortification of grain crops is considered the best method for increasing human Se intake, which may help people alleviate Se-deficiency. To investigate the efficiency of agronomic Se biofortification of oat, four Se fertilizer application treatments were tested: topsoil (T), foliar (S), the combination of T and S (TS), and control without Se application (CK). Compared with CK, TS significantly increased the 1000-grain weight, grain yield, Se contents in all parts of oats, contents of soil available N, K, and organic matter by 18%, 8.70%, 19.7-60.2%, 6.00%, 8.02%, and 17.95%, respectively. Leaves, roots, and ears had the highest conversion rate of exogenous Se in S (644.63%), T (416.00%), and TS (273.20%), respectively. TS also increased the activities of soil urease, alkaline phosphatase, and sucrose and the diversity of soil bacterial communities. TS and T increased the relative abundance of bacteria involved in the decomposition of organic matter, such as Actinobacteria, Gemmatimonadetes, Chloroflexi, and Bacteroidetes positively correlated with soil nutrients and enzyme activities, and reduced Proteobacteria and Firmicutes negatively correlated with them, Granulicella, Bacillus, Raoultella, Lactococcus, Klebsiella, and Pseudomonas. Furthermore, TS significantly increased the relative abundance of Planctomycetes, Chlorobi, Nitrospinae, Nitrospirae, Aciditeromonas, Gemmatimonas, Geobacter, and Thiobacter. T significantly increased the abundance of Lysobacter, Holophaga, Candidatus-Koribacter, Povalibacter, and Pyrinomonas. S did not significantly change the bacterial communities. Thus, a combined foliar and soil Se fertilizer proved conducive for achieving higher yield, grain Se content, and improving Se transport, the diversity of rhizosphere bacterial community, and bacterial functions in oats.