•Human thermal comfort (PET) under different tree species have been investigated.•Eco-physiological and bio-meteorological data were measured across the city.•Tree species with higher leaf area index showed better below-canopy surface cooling.•Along with higher tree transpiration, they provided better PET reduction.•Cluster tree planting design provided better PET even they reduced the tree growth. The potential of urban greenspaces in regulating temperature is widely known; however, in the heterogeneous urban landscape the process and the magnitude of human thermal comfort as well as the cumulative effects of natural and built surfaces are not yet well understood. We selected two ecologically contrasting species Tilia cordata Mill. and Robinia pseudoacacia L. planted in eight plots across five different sites in Würzburg, one of the major cities in Northern Bavaria, Germany. We monitored meteorological and edaphic variables and tree sap-flow along with the air temperature, relative humidity, within tree canopy and outside, between January 1 and December 31, 2018. Additionally, we measured surface temperature, meteorological variables below the tree canopies and outside, during the hottest summer days of 2018 (July 23 to 31). With 35% higher leaf area index, T. cordata tree canopies prevented higher incoming radiation compared to R. pseudoacacia trees. Moreover, with diffuse porous wood anatomy, T. cordata trees provided four times more transpiration compared to R. pseudoacacia, therefore, up to 2.8 °C air temperature reduction (ΔAT) within the tree canopy, and increase in absolute humidity (ΔAH) up to 2.6 g m − 3 compared to the 1.9 °C of ΔAT and 1.9 g m − 3 of ΔAH for R. pseudoacacia respectively. Thus, physiological equivalent temperature reduction was up to 4 °C under the shade of a R. pseudoacacia tree compared to 11 °C under a T. cordata tree. Finally, the research showed the importance of urban topography, such as street orientation, surrounding environment such as geometry, urban design to reduce the hindrance of wind flow and proportion of greenspaces in terms of outdoor human thermal comfort.