Urbanisation transforms natural environments, impacting not only wild animals living in cities but also the microorganisms they are hosting. To better understand urban-driven variation in microbiological composition and diversity in the gut of birds developing in urban areas, we collected faecal samples from blue tit Cyanistes caeruleus nestlings using nestboxes distributed across the capital city of Warsaw, Poland. Sampling included a variety of urban habitats, a suburban village and a natural forest area. Microbiome analysis revealed a pattern of reduced alpha diversity and significant shifts in beta diversity in urbanised settings, driven by impervious surface coverage. Additionally, we observed that this effect was year-dependent, therefore highlighting the importance of temporal replication in ecological research. Furthermore, comparing two cavity types (natural and human-made), we demonstrated that artificial nestboxes, a tool widely used in field ecology, can impact the microbiome assembly in nestlings.