Man-made environments such as ports and marinas are gateways for many marine non-indigenous species (mNIS) transported via shipping worldwide. These habitats are often the focus of biosecurity programs for the surveillance of mNIS, but there have been few studies investigating the distribution of biofouling communities in natural habitats and nearby artificial habitats. Here, following a DNA metabarcoding approach, we tested for differences in spatio-temporal trends of biological pioneer communities established after one-week colonization between a port, a marina, and coral reefs using two different matrices (PVC panels and water samples) in the central Red Sea. In addition, we aimed to investigate differences in the prevalence of mNIS between man-made and natural habitats during the summer and winter seasons, and whether the number and patterns of mNIS differed between the port (an active historic port subjected to intense shipping traffic) and the marina (a small recently developed marina utilized by small and medium research vessels). The community structure from samples collected at the two coral reefs showed greater similarity to each other compared to that obtained from the port and the marina. A total of 29 mNIS were detected across all sites, of which 16 were reported in the Red Sea for the first time, with Jeddah port hosting 28 mNIS. By applying standardized methodological approaches, this study provides comparable data regarding the monitoring of introduced and invasive species, particularly within the data-poor Red Sea, a major shipping corridor. This study serves as the foundation for implementing a robust and rapid baseline monitoring system to assess native biodiversity and facilitate the early detection of NIS.