Simple Summary : Nest establishment is a critical stage of the ant life cycle because it determines the chances of colony success. Here, we study the effect of different numbers of queens (i.e., one or six) on the position of queens and workers inside and outside the artificial nests of an invasive (Linepithema humile) and Mediterranean native (Tapinoma nigerrimum) species. Our results suggest that queens in nests with six queens entered the nest faster than single queens. Similarly, during nest establishment, workers in nests with six queens entered the nest faster, with this effect being more pronounced for the native species. Once nests were established, fewer workers were engaged in outside-nest tasks in nests with six queens. This was especially true for workers of the native species engaged in patrolling. These results suggest that the number of queens can influence both queen and worker behavior, and that invasive and native species have different responses. Abstract : As a critical stage in the life cycle of ant colonies, nest establishment depends on external and internal factors. This study investigates the effect of the number of queens on queen and worker behavior during nest establishment in invasive Argentine ants (Linepitema humile) and native Mediterranean Tapinoma nigerrimum. We set up experimental colonies with the same number of workers but with one or six queens. At different time points, we recorded the positions of queens and workers inside and outside the nest. Our results highlight the influence of the number of queens on the position of queens and workers with between-species differences. Queens of both species entered the nests more quickly when there were six queens. During nest establishment, more workers were inside nests with six queens for both species, with this effect being greater for T. nigerrimum. Once nests were established, fewer workers of both species were engaged in nest maintenance and feeding in nests with six queens; T. nigerrimum had fewer workers engaged in patrolling. These results suggest that the number of queens is a key factor driving queen and worker behavior during and after nest establishment with different species responses.