Hospitals represent the most energy-intensive buildings in most of the World, mainly because of strict requirements for micro-climatic control and high energy needs for equipment. Thus, the effective energy design and/or retrofit of these buildings can yield huge energy, environmental and economic benefits. In this frame, the study investigates the energy retrofit of a hospital building, i.e., the “D” Pavilion of the “A. Cardarelli” hospital facility in Naples (South Italy). The building is modeled in EnergyPlus and the energy model is calibrated by comparison against measured data, provided by an annual experimental campaign. Hence, in order to address building energy retrofit, a rigorous cost-optimal analysis is conducted by coupling EnergyPlus and MATLAB®. The investigated energy retrofit measures (ERMs) are addressed to the energy systems and to the exploitation of renewable energy sources. ERMs for the building envelope are excluded because they are not effective for the considered case study. The optimization allows to explore a comprehensive domain of retrofit scenarios, and thus, finally, robust cost-optimal solutions are found in correspondence of different discount rates. These solutions yield substantial reductions of primary energy consumption, global cost and polluting emissions with payback periods around 4 years.