Despite the critical ecological role of phytoplankton, knowledge of their community structure across the Indonesian archipelago remains limited. This study tested Wallace Line hypothesis by evaluating whether distinct marine phytoplankton assemblages exist across different biogeographic realms. We employed LSU D1–D2 rDNA metabarcoding to analyze surface-water samples from 96 stations collected between 2019 and 2025, covering the Indomalayan (Panjang), the Australasian (Manado and Tagulandang) regions, and the transitional zone (Lombok). Our results of alpha and beta diversity analyses revealed clear spatial segregation in community composition across the surveyed regions. Furthermore, we demonstrated that community assembly was independently shaped by both environmental filtering and spatially structured processes. We detected 67 harmful algal bloom (HAB) taxa, uncovering numerous new regional records for Indonesia, though only 23 species were cosmopolitan across all regions. The discovery of mixed taxonomic ribotypes representing Indomalayan and Australasian origins, including Alexandrium pacificum, A. minutum (ribotypes B1 and B2), Karlodinium australe, and Karenia cristata, strongly suggested that the Indonesian archipelago functions as a transregional dispersal corridor. Nutrient gradients and temperature-salinity contrasts emerged as major axes of ecological niche differentiation for the dominant HAB taxa. Our findings highlight how oceanographic connectivity and transregional transport influence HAB dynamics, emphasizing the need for integrated ecosystem monitoring and management in this ecologically and economically important region.