This study quantifies the environmental impact of milk production of Italian Mediterranean buffaloes and points out the farm characteristics that mainly affect their environmental performance. Life cycle assessment was applied in a sample of 6 farms. The functional unit was 1 kg of normalized buffalo milk (LBN), with a reference milk fat and protein content of 8.3 and 4.73%, respectively. The system boundaries included the agricultural phase of the buffalo milk chain from cradle to farm gate. An economic criterion was adopted to allocate the impacts on milk production. Impact categories investigated were global warming (GW), abiotic depletion (AD), photochemical ozone formation (PO), acidification (AC), and eutrophication (EU). The contribution to the total results of the following farm activities were investigated: (1) on-farm energy consumption, (2) manure management, (3) manure application, (4) on-farm feed production (comprising production and application of chemical fertilizers and pesticides), (5) purchased feed production, (6) enteric fermentation, and (7) transport of purchased feeds, chemical fertilizers, and pesticides from producers to farms. Global warming associated with 1 kg of LBN resulted in 5.07 kg of CO₂ Eq [coefficient of variation (CV)=21.9%], AD was 3.5 × 10(-3) kg of Sb Eq (CV=51.7%), PO was 6.8 × 10(-4) kg of C₂H₄ Eq (CV=28.8%), AC was 6.5 × 10(-2) kg of SO₂ Eq (CV=30.3%), and EU was 3.3 × 10(-2) kg of PO₄(3-) Eq (CV=36.5%). The contribution of enteric fermentation and manure application to GW is 37 and 20%, respectively; on-farm consumption, on-farm feed production, and purchased feed production are the main contributors to AD; about 70% of PO is due to enteric fermentation; manure management and manure application are responsible for 55 and 25% of AC and 25 and 55% of EU, respectively. Methane and N₂O are responsible for 44 and 43% of GW, respectively. Crude oil consumption is responsible for about 72% of AD; contribution of CH4 to PO is 77%; NH₃ is the main contributor to AC (92%); NO₃(-) and NH₃ are responsible for 55 and 41% of EU, respectively; contribution of P to EU is only 3.2%. The main characteristics explaining the significant variability of life cycle assessment are milk productivity and amount of purchased feed per kilogram of LBN. Improvement of LBN production per buffalo cow is the main strategy for reducing GW and PO; improvement of the efficiency of feed use is the strategy proposed for mitigating AD, PO, AC, and EU.