In the last decade a large collection of samples and associated metadata have been obtained from Australia’s Integrated Marine Observing System network of national reference stations and various research voyages around Australia as part of the Australian Marine Microbial Biodiversity Initiative (AMMBI). These samples are enabling the systematic evaluation of the diversity, abundance and distributions of Australian marine microbial communities on an unprecedented scale. Here we report the analysis of phytoplankton communities in the highly productive, tropical marine environments of northern Australia. From previous studies using primarily microscopic and pigment analyses these waters have been characterised as being dominated by large diatoms, dinoflagellates and the nitrogen fixing, marine cyanobacterium Trichodesmium. However, using a combination of Illumina-based 16S and 18S ribosomal RNA amplicon sequencing and flow cytometry on surface samples collected during an oceanographic transect from the Arafura Sea through the Torres Strait to the Coral Sea, we found that unicellular picocyanobacterial primary producers dominated the phytoplankton communities while picoeukaryotic phytoplankton formed a consistent, though smaller proportion. Major taxonomic groups displayed distinct biogeographic patterns. Unicellular picocyanobacteria dominated in both flow cytometric abundance and carbon biomass, with members of the Synechococcus genus dominating in the shallower Arafura Sea and Torres Strait, and Prochlorococcus dominating in the oligotrophic, low chlorophyll waters of the Coral Sea. Consistent with previous observations, sequence analysis indicated that a variety of diatoms exhibited high relative abundance in the Arafura Sea and Torres Strait, while dinoflagellates and prymnesiophytes were more abundant in the Coral Sea. Ordination analysis identified temperature, nutrients and water depth as environmental determinants of assemblage composition. Additionally, we found that the biggest contributor to the satellite-derived surface chlorophyll a signal was Synechococcus rather than the picoeukaryotic phytoplankton. Similar analyses of phytoplankton communities will be done on some of the other available AMMBI samples to better understand phytoplankton patterns in Australian marine waters and how they might respond to oceanic change now and in the future.