In this paper, multi-frequency synthetic aperture radar (SAR) data at L- and C-bands (ALOS PALSAR and Envisat/ASAR) were used to estimate forest biomass in Tuscany, in Central Italy. The ground measurements of woody volume (WV, in m3/ha), which can be considered as a proxy of forest biomass, were retrieved from the Italian National Forest Inventory (NFI). After a preliminary investigation to assess the sensitivity of backscatter at C- and L-bands to forest biomass, an approach based on an artificial neural network (ANN) was implemented. The ANN was trained using the backscattering coefficient at L-band (ALOS PALSAR, HH and HV polarization) and C-band (Envisat ASAR in HH polarization) as inputs. Spatially distributed WV values for the entire test area were derived by the integration (fusion) of a canopy height map derived from the Ice, Cloud, and Land Elevation Geoscience Laser Altimeter System (ICESat GLAS) and the NFI data, in order to build a significant ground truth dataset for the training stage. The analysis of the backscattering sensitivity to WV showed a moderate correlation at L-band and was almost negligible at C-band. Despite this, the ANN algorithm was able to exploit the synergy of SAR frequencies and polarizations, estimating WV with average Pearson’s correlation coefficient (R) = 0.96 and root mean square error (RMSE) ≃ 39 m3/ha when applied to the test dataset and average R = 0.86 and RMSE ≃ 75 m3/ha when validated on the direct measurements from the NFI. Considering the heterogeneity of the scenario (Mediterranean mixed forests in hilly landscape) and the small amount of available ground measurements with respect to the spatial variability of different plots, the obtained results can be considered satisfactory. Moreover, the successful use of WV from global maps for implementing the algorithm suggests the possibility to apply the algorithm to wider areas or even to global scales.