The increasing demand for renewable energy, coupled with the urgent challenges posed by climate change, has positioned perennial biomass crops (BPGs) as essential and sustainable alternatives for bioenergy production. This study investigated the impact of irrigation regimes on the physiological performance of three BPG species-Arundo donax L., Saccharum spontaneum, and Miscanthus-with a focus on leaf gas exchange (net assimilation rate and transpiration rate) and instantaneous water use efficiency (iWUE) at varying levels of irrigation input, adopting a split-plot experimental design under the Mediterranean climatic conditions of Sicily (Italy). The results clearly showed that A. donax, a C3 species, outperformed the C4 species S. spontaneum and Miscanthus, exhibiting significantly higher stomatal conductance and net photosynthesis, especially under irrigated conditions. S. spontaneum demonstrated the highest iWUE, particularly in rainfed treatments, reflecting its efficient use of water. Miscanthus showed the greatest sensitivity to water stress, with a more pronounced decline in photosynthesis during drought periods. This study accentuated the role of effective water management and genotype selection in optimizing biomass yield and resource efficiency, providing valuable insights for improving crop productivity in Mediterranean and other semi-arid regions.