Leaf biochemical traits indicating early symptoms of plant stress can be assessed using imaging spectroscopy combined with radiative transfer modelling (RTM). In this study, we assessed the potential applicability of the leaf radiative transfer model Fluspect-Cx to simulate optical properties and estimate leaf biochemical traits through inversion of two native Australian eucalypt species: Eucalyptus dalrympleana and E. delegetensis. The comparison of measured and simulated optical properties revealed the necessity to recalibrate the refractive index and specific absorption coefficients of the eucalypt leaves’ biochemical constituents. Subsequent validation of the modified Fluspect-Cx showed a closer agreement with the spectral measurements. The average root mean square error (RMSE) of reflectance, transmittance and absorptance values within the wavelength interval of 450–1600 nm was smaller than 1%. We compared the performance of both the original and recalibrated Fluspect-Cx versions through inversions aiming to simultaneously retrieve all model inputs from leaf optical properties with and without prior information. The inversion of recalibrated Fluspect-Cx constrained by laboratory-based measurements produced a superior accuracy in estimations of leaf water content (RMSE = 0.0013 cm, NRMSE = 6.55%) and dry matter content (RMSE = 0.0036 g·cm−2, NRMSE = 21.28%). The estimation accuracies of chlorophyll content (RMSE = 8.46 µg·cm−2, NRMSE = 24.73%), carotenoid content (RMSE = 3.83 µg·cm−2, NRMSE = 30.82%) and anthocyanin content (RMSE = 1.69 µg·cm−2, NRMSE = 37.12%) were only marginally better than for the inversion without any constraints. Additionally, we investigated the possibility to substitute the prior information derived in the laboratory by non-destructive reflectance-based spectral indices sensitive to the retrieved biochemical traits, resulting in the most accurate estimation of carotenoid content (RMSE = 3.65 µg·cm−2, NRMSE = 29%). Future coupling of the recalibrated Fluspect with a forest canopy RTM is expected to facilitate retrieval of biophysical traits from spectral air/space-borne image data, allowing for assessing the actual physiological status and health of eucalypt forest canopies.