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The present research investigates through metagenomics sequencing the unicellular protistan communities in Thermaikos Gulf. This research analyzes the diversity, composition and abundance in this marine environment. Water samples were collected monthly from April 2017 to February 2018 in the port of Thessaloniki (Harbor site, 40o 37’ 55 N, 22o 56’ 09 E). The extraction of DNA was completed as well as the sequencing was performed, before the downstream read processing and the taxonomic classification that was assigned using PR2 database. A total of 1248 Operational Taxonomic Units (OTUs) were detected but only 700 unicellular eukaryotes were analyzed, excluding unclassified OTUs, Metazoa and Streptophyta. In this research-based study the most abundant and diverse taxonomic groups were Dinoflagellata and Protalveolata. Specifically, the most abundant groups of all samples are Dinoflagellata with 190 OTUs (27.70%), Protalveolata with 139 OTUs (20.26%) Ochrophyta with 73 OTUs (10.64%), Cercozoa with 67 OTUs (9.77%) and Ciliophora with 64 OTUs (9.33%). The most diverse taxonomic groups are Dinoflagellata with 109508 reads, Protalveolata with 21977 reads and Ochrophyta with 11338 reads. At parallel, some of the dominant OTUs are Akashiwo with 47673 reads Noctiluca with 18025 reads and Syndiniales Group I with 14723 reads. The most abundant samples occurred to be the samples of 9/5/17 with 208 OTUs, 13/12/17 with 202 OTUs, 23/8/17 with 201 OTUs and 26/7/17 with 196 OTUs, while the least abundant are the samples of 15/11/17 with 93 OTUs, 18/10/17 with 101 OTUs, 6/2/18 with 109 OTUs and 28/6/17 with 124 OTUs. The highest number of OTUs from the Closest Relatives (Table 6) are Ptychodiscus noctiluca, Protodinium simplex, Biecheleriopsis adriatica, Protoperidinium pellucidum, Gyrodinium cf. gutrula and Micromonas pusilla. This study will provide data for future metagenomics studies and will contribute for a better understanding about the unicellular protistan communities in Thermaikos Gulf.