Spatial predictions of biomass production and biodiversity at regional scale in grasslands are critical to evaluate the effects of management practices across environmental gradients. New generations of remote sensing sensors and machine learning approaches can predict these grassland characteristics with varying accuracy. However, such studies frequently fail to cover a sufficiently broad range of environmental conditions, and their prediction models are often case-specific. To address this gap, we have modelled above-ground biomass and species richness in 150 spatially independent grassland plots of three geographical regions in Germany. These regions follow a North-South climate gradient and differ in soil types, topography, elevation, climatic conditions, historical contexts, and management intensities. The predictors tested in this study are Sentinel-1 backscatter, Sentinel-2 time series of surface reflectance along with derived vegetation indices and Rao's Q, and a set of topoedaphic variables. We compared the performance of a feed-forward deep neural network (DNN) with a random forest (RF) regression algorithm. The DNN achieved the best estimations of biomass (r2 = 0.45) when trained with Sentinel-2 surface reflectance only. Moreover, the DNN showed a higher generalizability than RF during spatial cross-validations (i.e., calibrating and validating in different regions, r2 = 0.38 vs. 0.26). Species richness predictions by both algorithms improved when the full time series of Sentinel-2 surface reflectance values were used (highest r2 = 0.42 achieved by the DNN), but both performed poorly during spatial cross-validations. Overall, the DNN-based models were more robust than RF models, showed a lower bias and lower systematic error, and required fewer inputs. Explainability analysis indicated that red-edge and near infrared information from May and October was the most relevant to predict species richness. This study presents an important step forward in generating robust spatially explicit predictions of grassland attributes and biodiversity variables across large areas, environmental gradients, and phenological stages.