Forests play a crucial role in maintaining ecological balance and biodiversity, making the accurate mapping of tree species and assessment of biodiversity indices essential for informed management decisions. This study introduces an innovative methodology that integrates EnMAP (Environmental Mapping and Analysis Program) hyperspectral data with Sentinel-2 multitemporal data to classify tree species in the biodiverse landscapes of Kampinos National Park and its surrounding regions in Poland.We extract essential vegetation indices such as NDVI, NDMI, SAVI, and EVI from Sentinel-2 data to assess forest health and dynamics. The Sentinel-2 data is upscaled from 10 m to 30 m to align with EnMAP’s spatial resolution, followed by precise co-registration of the images using QGIS. Utilizing a rich dataset from the National Forest Inventory, we extract spectral signatures of nine distinct tree species from both data sources. We employ five machine learning algorithms—Support Vector Machines (SVM), Random Forest (RF), CatBoost (CAT), Gradient Boosting Classifier (GBC), and XGBoost (XGB)—to enhance classification accuracy.Through iterative experimentation with data reduction techniques and algorithm tuning, we achieve optimal performance across needle-leaved and broad-leaved species. The resulting tree species maps are validated through quantitative accuracy assessments against mixed-species polygons from the National Forest Inventory and ground truthing in the Kampinos National Park. Achieving an overall accuracy of 85% to 93%, our study demonstrates the efficacy of this integrated approach in tree species mapping. Furthermore, the tree species maps serve as a foundation for deriving key biodiversity indices—species richness, Shannon-Wiener Diversity Index, Simpson’s Diversity Index, and a composite Biodiversity Index—providing insights into spatial biodiversity patterns and informing targeted conservation strategies. This study exemplifies the potential of combining advanced remote sensing techniques with field validation to enhance our understanding of forest ecosystems and guide sustainable management practices.