The assessment of mature urban tree vitality using physiological measurements is still in its infancy. Chlorophyll fluorescence is a method for assessing tree vitality that has potential for use in urban environments, particularly on trunk bark, which is easy to access from the ground. Here we describe how we compared bark and leaf fluorescence in a variety of street and park trees (Ficus macrophylla Pers., Platanus x acerifolia (Aiton) Willd., and Ulmus parvifolia Jacq.) with pre-dawn water potential as a way of determining the cause of potential physiological stress in the summer of 2012. Statistical relationships were observed between bark chlorophyll fluorescence and pre-dawn water potential in Ficus macrophylla and Platanus x acerifolia, but were not as consistent in Ulmus parvifolia. In addition, bark and leaf chlorophyll fluorescence were compared with an urban visual vitality index both in autumn 2011 and summer 2012. In this case statistical relationships between bark chlorophyll fluorescence values and urban tree visual vitality were almost non-existent in the Ficus macrophylla and Platanus x acerifolia trees, however, statistical relationships were significant between bark chlorophyll fluorescence and the urban tree vitality index in Ulmus parvifolia. Bark chlorophyll fluorescence may become a useful tool for measuring physiological stress in trees, but further work needs to be undertaken to clarify and better understand the varying responses of different tree species.