A significant trend has developed with the recent growing interest in the estimation of aboveground biomass of vegetation in legume-supported systems in perennial or semi-natural grasslands to meet the demands of sustainable and precise agriculture. Unmanned aerial systems (UAS) are a powerful tool when it comes to supporting farm-scale phenotyping trials. In this study, we explored the variation of the red clover-grass mixture dry matter (DM) yields between temporal periods (one- and two-year cultivated), farming operations [soil tillage methods (STM), cultivation methods (CM), manure application (MA)] using three machine learning (ML) techniques [random forest regression (RFR), support vector regression (SVR), and artificial neural network (ANN)] and six multispectral vegetation indices (VIs) to predict DM yields. The ML evaluation results showed the best performance for ANN in the 11-day before harvest category (R² = 0.90, NRMSE = 0.12), followed by RFR (R² = 0.90 NRMSE = 0.15), and SVR (R² = 0.86, NRMSE = 0.16), which was furthermore supported by the leave-one-out cross-validation pre-analysis. In terms of VI performance, green normalized difference vegetation index (GNDVI), green difference vegetation index (GDVI), as well as modified simple ratio (MSR) performed better as predictors in ANN and RFR. However, the prediction ability of models was being influenced by farming operations. The stratified sampling, based on STM, had a better model performance than CM and MA. It is proposed that drone data collection was suggested to be optimum in this study, closer to the harvest date, but not later than the ageing stage.