Regulatory constraints and environmental and human health concerns have promoted the search for alternative bio-control strategies of fire blight, a destructive disease of rosaceous plants which produces serious losses in apple and pear orchards all over the world. The aim of this study was to establish the antimicrobial activity of Citrus maxima essential oil against Erwinia amylovora. An agar diffusion method was used for the screening of the inhibitory effect of Citrus maxima essential oil on bacterial strains growth. The quantitative inhibitory effect of pomelo oil on in vitro biofilm development was established by a microtiter colorimetric assay. In order to investigate the ability of pomelo oil to interfere with bacterial adherence and subsequent biofilm development on leaves obtained from different pomaceous fruit trees species and cultivars: Pyrus (Napoca, Williams), Malus (Golden Delicious) and Cydonia (Aromate), leaves were immersed in pomelo oil for 1, 2, 3, 5 and 10 minutes before exposing them to bacterial colonization. The architecture of bacterial biofilms developed on leaf surface was analyzed using Confocal Scanning Laser Microscopy (CSLM). Our results showed that Citrus maxima essential oil inhibited the development of bacterial biofilms on leaves, pomelo oil being more active on Cydonia (Aromate) leaves when the leaves were treated for 5 minutes. The results obtained from this study may contribute to the development of new bio-control agents as alternative strategies to protect fruit trees from fire blight disease.