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The introduction of non-indigenous species (NIS) induces severe impacts on marine biodiversity and ecosystems. Macaronesia is an ecologically relevant region where several NIS were detected recently. For the first time, a standard experimental approach was designed to examine biofouling assemblages and investigate NIS across the region. In this context, sessile biofouling assemblages were examined in four recreational marinas in all the Macaronesian archipelagos from 2018 to 2020: the Azores, Madeira, Canary Islands, and Cabo Verde. We hypothesised that NIS numbers, abundance, and recruitment differed in each location due to abiotic and biotic features. From the Azores (higher latitudes) to Cabo Verde (lower latitudes), NIS recruitment and percentage cover decreased following a partial latitude gradient. The present study unveiled 25 NIS, with new records for the Azores (two cryptogenic species), Canary Islands (one NIS and two cryptogenic species), and Cabo Verde (three NIS and three cryptogenic species). The present research represents a pioneer and relevant step in advancing our current understanding of marine biological invasions in Macaronesia, employing a standard and low-cost approach. [Display omitted] • First experimental study on NIS in the Macaronesia • NIS recruitment and percentage cover decreased with latitude. • New records for the Azores, Canary Islands and Cabo Verde • Advancing our current understanding of marine biological invasions in Macaronesia [ABSTRACT FROM AUTHOR]