The invasion of the Argentine ant, Linepithema humile (Mayr) (Hymenoptera, Formicidae) can alter the entire ecosystem with serious impacts on the native community structure (e.g., ant diversity) and processes (e.g., trophic interactions) leading to biodiversity loss and pest outbreaks. Most studies addressing these impacts have been conducted in natural or semi-natural areas, few are those conducted in agricultural ecosystems, such as citrus orchards. These are dominant agricultural ecosystems in Mediterranean landscapes. Furthermore, most studies have been conducted in a short span, not evidencing seasonal fluctuations. In this work, we assessed the ecological impact of the Argentine ant on the native ant communities in citrus orchards, in the region of Algarve, southern Portugal. By using principal response curve, we compared seasonal variation on ant assemblages in invaded and uninvaded citrus orchards foraging on tree canopy from a two-year sampling. The Argentine ant had a marked negative impact on the native ant community foraging on citrus canopy. In the uninvaded orchards, the native ant community had a rich assemblage composed of 16 ant species, in its majority (72%) controlled by the dominant species Lasius grandis Forel, Tapinoma nigerrimum (Nylander) and/or Pheidole pallidula (Nylander). In the invaded orchards, the native ant community was poorer and highly modified, mostly dominated by the Argentine ant (80%). Apparently, the only native ant species not affected by the presence of the Argentine ant was Plagiolepis pygmaea (Latreille). A significant negative effect was found between the proportion of infested trees by L. humile and the number of native ant species per orchard. Differences in the native ant community in the invaded and uninvaded orchards persisted over seasons and years. However, negative impacts were higher in the spring and summer, and less pronounced in the autumn. We discuss implications for citrus pest management.