Urbanisation has reduced the abundance and diversity of many taxonomic groups, and the effects may be more pronounced on islands, which have a smaller regional species pool to compensate. Green spaces within urban environments may help to safeguard wildlife assemblages, and the associated habitat heterogeneity can even increase species diversity. Here, total abundance and species diversity of butterflies, birds, and vegetation at nine rural and nine urban locations were quantified on Lipsi Island, Greece. Sites were assessed using Pollard walks for butterflies, point-count surveys for birds, and quadrats for vegetation. There was no significant difference in the abundance or species diversity of butterflies or vegetation among rural and urban locations, which could pertain to the low building density within urbanised areas and the minimal extent of urbanisation on the island. However, urban areas hosted a significantly greater abundance and richness of birds compared to rural sites. The community composition of butterflies, birds, and vegetation also differed significantly between urban and rural locations, highlighting the impact of urbanisation on species across a broad range of trophic groups. This study contributes to ecological knowledge on the impacts of urbanisation across multiple trophic levels in island ecosystems, with comparisons across a gradient of island size and urbanisation intensity needed in future research.