Urban street trees provide many environmental, social, and economic benefits for our cities. This research explored the role of street trees in Melbourne, Australia, in cooling the urban microclimate and improving human thermal comfort (HTC). Three east–west (E–W) oriented streets were studied in two contrasting street canyon forms (deep and shallow) and between contrasting tree canopy covers (high and low). These streets were instrumented with multiple microclimate monitoring stations to continuously measure air temperature, humidity, solar radiation, wind speed and mean radiant temperature so as to calculate the Universal Thermal Climate Index (UTCI) from May 2011 to June 2013, focusing on summertime conditions and heat events. Street trees supported average daytime cooling during heat events in the shallow canyon by around 0.2 to 0.6 °C and up to 0.9 °C during mid-morning (9:00–10:00). Maximum daytime cooling reached 1.5 °C in the shallow canyon. The influence of street tree canopies in the deep canyon was masked by the shading effect of the tall buildings. Trees were very effective at reducing daytime UTCI in summer largely through a reduction in mean radiant temperature from shade, lowering thermal stress from very strong (UTCI > 38 °C) down to strong (UTCI > 32 °C). The influence of street trees on canyon air temperature and HTC was highly localized and variable, depending on tree cover, geometry, and prevailing meteorological conditions. The cooling benefit of street tree canopies increases as street canyon geometry shallows and broadens. This should be recognized in the strategic placement, density of planting, and species selection of street trees.