•Mismatch of Regulating Ecosystem Services varies considerably in the city structure.•Urban planning strategies generally do not consider the Ecosystem Services mismatch.•Mapping priority areas helps to exploit the multifunctionality of Ecosystem Services.•Green Infrastructure also provides important economic benefits to the society.•Targeted urban planning improves human health and well-being. Balancing the ecosystem service (ES) mismatch should be a goal of sustainable urban planning. However, (i) many urban areas lack an assessment of this mismatch and (ii) scientific findings are not easily translatable into good practices. In this study, we assessed the mismatch for two regulating ESs—regulation of air quality (intended as PM10 removal by vegetation) and urban temperature regulation—in the Municipality of Rome (Italy). The spatial distribution of the ES mismatch was then used to identify priority intervention areas (PIAs), namely those that would benefit the most from targeted urban planning. To do so, we computed composite indicators of supply and demand for each ES, adopting a process-based approach. Additionally, a monetary valuation of the related benefits associated with urban green infrastructure is provided. Our findings suggest that regulation of air quality falls short in highly urbanized areas, whereas a mismatch in urban temperature regulation is observed in both highly urbanized areas and some agricultural lands. The majority of the PIAs fall in the eastern and southern sectors of the Municipality of Rome. Our findings also indicate that urban planning should consider urban regeneration practices and reforestation of existing green areas within the PIAs. Sustainable urban planning can produce remarkable environmental benefits, as estimated, for the Municipality of Rome, up to several hundred million euros per year, depending on the methodology used for the monetary valuation.