The uses of precision oliviculture have increased in recent years to improve the quality and quantity of extra virgin olive oil. In traditional and intensive systems, biennial pruning is often applied to balance and maintain plant vigour, aiming at reducing management costs. This study presents the results of a three-year experiment with the objective of quantifying the effects of biennial pruning on the vegetative vigour of olive trees, investigating the geometric and spectral characteristics of each canopy determined with multispectral images acquired by UAV. The experiment was carried out in an olive orchard located in western Sicily (Italy). Multispectral images were acquired using a UAV in automatic flight configuration at an altitude of 70 m a.g.l. The segmentation and classification of the images were performed using Object-Based Image Analysis (OBIA) based on the Digital Elevation Model (DEM) and orthomosaic to extract the canopy area, height, volume and NDVI for each plant. This study showed that the technology and image analysis processing used were able to estimate vigour parameters at different canopy densities, compared to field measurements (R2 = 0.97 and 0.96 for canopy area and volume, respectively). Furthermore, it was possible to determine the amount of removed biomass for each plant and vigour level. Biennial pruning decreased the number of plants initially classified as LV (low-vigour) and maintained a vegetative balance for MV (medium-vigour) and HV (high-vigour) plants, reducing the spatial variability in the field.