Abstract Population size estimation is performed for several reasons including disease surveillance and control, for example to design adequate control strategies such as vaccination programs or to estimate a vaccination campaign coverage. In this study, we aimed at assessing the benefits and challenges of using Unmanned Aerial Vehicles (UAV) to estimate the size of free-roaming domestic dog (FRDD) populations and compare the results with two regularly used methods for population estimations: a Bayesian statistical model based on capture-recapture data and the human:dog ratio estimation. Three studies sites of one square kilometer were selected in Petén department, Guatemala. UAV flight were conducted twice during two consecutive days per study site. The UAV’s camera was set to regularly take pictures and cover the entire surface of the selected areas. A door-to-door survey was conducted in the same areas, all available dogs were marked with a collar and owner were interviewed. Simultaneously to the UAV’s flight, transect walks were performed and the number of collared and non-collared dogs were recorded. Data collected during the interviews and the number of dogs counted during the transect walks informed a Bayesian statistical model. The number of dogs counted on the UAV’s pictures and the estimates given by the Bayesian statistical model, as well as the estimates derived from using a 5:1 human:dog ratio were compared to dog census data. FRDD could be detected using the UAV’s method. However, the method lacked of sensitivity, which could be overcome by choosing the flight timing and the study area wisely, or using infrared camera or automatic detection of the dogs. We also suggest to combine UAV and capture-recapture methods to obtain reliable FRDD population size estimated. This publication may provide helpful directions to design dog population size estimation methods using UAV.