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12 pages, 7 figures, 4 tables.-- Under a Creative Commons license, 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, NC is funded by a doctoral grant (SFRH/BD/146881/2019) awarded by Fundação para a Ciência e a Tecnologia (FCT). PR is financed by project MARE - Centro de Ciências do Mar e do Ambiente (UIDB/04292/2020). IG is supported financially by a Maria Zambrano contract UCA under the grants call for the requalification of the Spanish university system 2021–2023, funded by the European Union – NextGenerationEU. AC and MP had the support of FEDER funds through the Operational Programme for Competitiveness Factors -COMPETE and by National Funds through FCT under the UID/BIA/50027/2020 and POCI-01-0145-FEDER-006821. JCC is funded by FCT under the Scientific Employment Stimulus - Institutional Call - (CEECINST/00098/2018). This work was partially funded by MIMAR+ (MAC2/4.6.d/249) in the INTERREG MAC 2014-2020 Programme framework. This study had the support of FCT through the strategic project UIDB/04292/2020, awarded to MARE and through project LA/P/0069/2020, granted to the Associate Laboratory ARNET. This is contribution 129 from the Smithsonian's MarineGEO and Tennenbaum Marine Observatories Network