Agricultural intensification and soil mismanagement have been recognized among the main causes of soil erosion in Mediterranean climate areas such as the Arbia stream basin (Tuscany, Italy). This study aims at predicting soil loss from agricultural fields as it is essential for providing reliable information for prioritizing soil conservation measures. Thus, measured soil loss from 243 agricultural fields within the Arbia stream basin during the period 2007–2010 were used to calibrate and validate the ArcSWAT 2012 model at hydrological response units (HRU) scale. Analysis of variance with post-hoc Tukey honest significant test was used to assess significant measured soil loss differences between slope steepness classes and land covers. Soil loss estimation was always “very good” for irrigated field crops, olive groves, and vineyards, “good” for unirrigated field crops, and “unsatisfactory” for broad-leaved forest. The model succeeded in the quantitative assessment of erosive processes at HRU scales. Its application to the whole Arbia stream basin estimated that 31% of the total surface is subjected to higher erosion levels. This approach might help facilitate the identification of priority areas that need the implementation of conservation measures.