Street trees are integral components of urban green infrastructure. The importance of benefits provided by street trees has motivated the development of various tools to quantify the value of ecosystem services. The i-Tree Eco is a widely applied method for quantifying urban forest structure, ecosystem services, and values. Since its first release in 2006, i-Tree Eco has been successfully utilized in over 100 countries around the world. This study described one of the first applications of the i-Tree Eco international project in Kyoto, Japan, by customizing the models and parameters to enhance the accuracy of analysis results. Kyoto’s street trees are prominently dominated by Ginkgo (Ginkgo biloba L.), Trident Maple (Acer buergerianum Miq.), Japanese Zelkova (Zelkova serrata (Thunb.) Makino.), Tuliptree (Liriodendron tulipifera L.), Flowering dogwood (Cornus florida L.), London Planetree (Platanus × acerifolia), Plum/cherry (Prunus spp.), and Weeping willow (Salix babylonica), which account for 92% of the 1230 sample trees and deliver ecosystem service benefits at US$71,434.21 annually or US$58.07/tree/year. The annual value of each function was estimated at US$41.34/tree for carbon storage and sequestration, US$3.26/tree for stormwater runoff reduction, US$11.80/tree for adverse health mitigation effects, and US$1.67/tree for energy savings. The street tree species of Kyoto city that produce the highest average annual benefits are among the largest trees currently in the population, including P. × yedoensis (US$225.32/tree), Z. serrata (US$123.21/tree), S. babylonica (US$80.10/tree), and P. × acerifolia (US$65.88/tree). Our results demonstrated a comprehensive understanding of street trees benefits for Kyoto city, providing baseline information for decision-makers and managers to make effective urban trees management decisions, developing policy, and setting priorities.