The Aquatina Lagoon (Southern Adriatic Sea, Mediterranean Ecoregion) is a transitional water ecosystem with great ecological and socio-economic interest. Anthropogenic activities around the lagoon (e.g., agriculture and tourism) and hydrology can affect the environmental quality and biodiversity of the lagoon. Herein, the dynamics and diversity of phytoplankton communities were studied before and after the opening of a new canal connecting the lagoon with the sea, by using different approaches based on an evaluation of the size and structure of the phytoplankton as well as the taxonomic analyses. The lagoon depicted time-related fluctuations in chemical-physical parameters. The phytoplankton trend was characterized by an increase in abundance and biomass in summer, when pico-sized autotrophs dominated. Generally, nano-sized phytoflagellates dominated the community, while micro-sized dinoflagellates and diatoms were less abundant. An increase in the phytoplankton taxa number was observed throughout the years. All the analyzed parameters were generally relatively homogeneous before the opening of the channel, while some quantitative differences among stations were observed in the second sampling period. Considering the statistical evidence, both environmental and biological parameters were affected by the “dilution” effect exerted by marine water inputs. This research supports the evidence that phytoplankton is a good indicator of the environmental status, and the obtained results contribute to the implementation of management strategies for the conservation of transitional water ecosystems.