Improving our understanding of the functions and processes of soil microbial communities and their interactions with the physicochemical soil environment requires large amounts of timely and cost-efficient soil data, which is difficult to obtain with routine laboratory-analytical methods. Soil spectroscopy with portable visible-to-near infrared (VNIR) and mid-infrared (MIR) instruments can fill this gap by facilitating the rapid acquisition of biotic and abiotic soil information. In this study, we evaluated the capabilities of VNIR and MIR spectroscopy to analyze soil physicochemical and microbial properties in a long-term grassland biodiversity-ecosystem functioning experiment. Soil samples were collected at the Jena Experiment (Jena, Germany) and measured with portable VNIR and MIR spectrometers in field-moist condition to determine their potential for on-site data collection and analysis. Reference data to calibrate spectroscopic models were acquired with routine analytical methods, including PLFA extractions of microbial biomarkers. We further collected reference VNIR and MIR data on pre-treated soils (dried and finely ground) to assess the anticipated impact of field measurements on spectroscopic calibrations. MIR spectra allowed more accurate estimates of soil physicochemical and microbial properties than VNIR data on pre-treated samples. For soils in field condition, MIR calibrations were more accurate for physicochemical properties, but VNIR data gave significantly better estimates of microbial properties. Combined VNIR/MIR estimates achieved the most accurate estimation results for all soil properties in each case. Soil physicochemical properties could be estimated from VNIR/MIR data with high accuracy (R2 = 0.72– 0.99) on pre-treated soil samples, whereas the results for soil microbial properties were more moderate (R2 = 0.66– 0.72). On field-moist soils, estimation accuracies decreased notably for organic and inorganic carbon (