Urban and peri-urban trees in major cities provide a gateway for exotic pests and diseases (hereafter “pests”) to establish and spread into new countries. Consequently, they can be used as sentinels for early detection of exotic pests that could threaten commercial, environmental and amenity forests. Biosecurity surveillance for exotic forest pests relies on monitoring of host trees — or sentinel trees — around high-risk sites, such as airports and seaports. There are few publicly available spatial databases of urban street and park trees, so locating and mapping host trees is conducted via ground surveys. This is time-consuming and resource-intensive, and generally does not provide complete coverage. Advances in remote sensing technologies and machine learning provide an opportunity for semi-automation of tree species mapping to assist in biosecurity surveillance. In this study, we obtained high resolution (≥12 cm), 10-band, multispectral imagery using the ArborCam™ system mounted to a fixed-wing aircraft over Sydney, Australia. We mapped 630 Pinus trees and 439 Platanus trees on-foot, validating their exact location on the airborne imagery using an in-field mapping app. Using a machine learning, convolutional neural network workflow, we were able to classify the two target genera with a high level of accuracy in a complex urban landscape. Overall accuracy was 92.1% for Pinus and 95.2% for Platanus, precision (user’s accuracy) ranged from 61.3% to 77.6%, sensitivity (producer’s accuracy) ranged from 92.7% to 95.2%, and F1-score ranged from 74.6% to 84.4%. Our study validates the potential for using multispectral imagery and machine learning to increase efficiencies in tree biosecurity surveillance. We encourage biosecurity agencies to consider greater use of this technology.