As the urban heat island effect has become a worldwide phenomenon commonly affecting densely built-up areas, public administrations need efficient strategies to mitigate its impact on human well-being and public health. The aim of this study was to define a replicable method to estimate the ecosystem services provided by public street trees as a supporting tool in the decision-making process of urban greenery management. We compared three street arrangements characteristic of a residential district in Pisa, Italy: (1) with large trees, (2) with small trees, and (3) without trees. First, the software i-Tree Eco was used to assess the benefits of public trees located in the case-study area when provided with the three scenarios. Second, the comparison was held on the field, and we collected data with a wet bulb globe temperature meter in order to evaluate the differences in pedestrian thermal comfort among the street arrangements. The results confirmed the importance of urban vegetation, as it has major impacts on carbon sequestration and storage, pollution removal, air humidity and quality, and shade, given bigger trees and canopy sizes. The loss of ecosystem services compared to the presence of large trees varied between 40% and 50% (no trees) and 30% and 40% (small trees).