Accurate and timely monitoring of biomass in breeding nurseries is essential for evaluating plant performance and selecting superior genotypes. Traditional methods for phenotyping above-ground biomass in field conditions requires significant time, cost, and labor. Unmanned Aerial Vehicles (UAVs) offer a rapid and non-destructive approach for phenotyping multiple field plots at a low cost. While Vegetation Indices (VIs) extracted from remote sensing imagery have been widely employed for biomass estimation, they mainly capture spectral information and disregard the 3D canopy structure and spatial pixel relationships. Addressing these limitations, this study, conducted in 2020 and 2021, aimed to explore the potential of integrating UAV multispectral imagery-derived canopy spectral, structural, and textural features with machine learning algorithms for accurate oat biomass estimation. Six oat genotypes planted at two seeding rates were evaluated in two South Dakota locations at multiple growth stages. Plot-level canopy spectral, structural, and textural features were extracted from the multispectral imagery and used as input variables for three machine learning models: Partial Least Squares Regression (PLSR), Support Vector Regression (SVR), and Random Forest Regression (RFR). The results showed that (1) in addition to canopy spectral features, canopy structural and textural features are also important indicators for oat biomass estimation; (2) combining spectral, structural, and textural features significantly improved biomass estimation accuracy over using a single feature type; (3) machine learning algorithms showed good predictive ability with slightly better estimation accuracy shown by RFR (R2 = 0.926 and relative root mean square error (RMSE%) = 15.97%). This study demonstrated the benefits of UAV imagery-based multi-feature fusion using machine learning for above-ground biomass estimation in oat breeding nurseries, holding promise for enhancing the efficiency of oat breeding through UAV-based phenotyping and crop management practices.