In the Mediterranean region, the expansion of greenhouse horticulture has enabled the year-round supply of fresh vegetables. Compared to open field horticulture, this farming method can generate higher returns for farmers. However, it is often associated with significant environmental pressures. This research aims to pinpoint important opportunities for improvement of the environmental and economic performance of greenhouse farming in the Mediterranean region by showing the life cycle’s environmental and economic impacts and by highlighting life cycle hotspots. This is achieved through the combined application of life cycle assessment and life cycle costing to four case studies (commercial greenhouses) spanning the Mediterranean Basin (Italy, Spain, Tunisia, and Turkey). The case study findings highlight the following environmental hotspots and related impacts: (i) fertigation management can generate up to 11,283 m3/ha/year of water use impact; (ii) fertilizer leaching can generate up to 27 kg of N eq marine eutrophication impact; and (iii) crop protection treatments can generate up to 130,037 kg 1,4-DCB of terrestrial ecotoxicity impact. The large use of plastic materials (greenhouse and fertigation infrastructures) is an additional critical aspect due to manufacturing and disposal, contributing to eutrophication impact categories. Economic hotspots are related to greenhouse management (up to 35% total costs of production) and hired labor (up to 40% total costs of production). The lessons learnt from these case studies offer valuable insights into the sustainability challenges of greenhouse horticulture across the Mediterranean region. The hotspot analysis points to the need for targeted interventions to mitigate the most critical impacts while ensuring economic viability. This study enriches scientific understanding by examining different production and socioeconomic contexts, offering crucial insights for the advancement of sustainable practices in greenhouse agriculture such as the use of decision support systems to optimize input use.