Within the framework of the European INSEA project, an advanced assimilation system has been implemented for the Pagasitikos Gulf ecosystem. The system is based on a multivariate sequential data assimilation scheme that combines satellite ocean sea color (chlorophyll-a) data with the predictions of a three-dimensional coupled physical-biochemical model of the Pagasitikos Gulf ecosystem presented in a companion paper. The hydrodynamics are solved with a very high resolution (1/100°) implementation of the Princeton Ocean Model (POM). This model is nested within a coarser resolution model of the Aegean Sea which is part of the Greek POSEIDON forecasting system. The forecast of the Aegean Sea model, itself nested and initialized from a Mediterranean implementation of POM, is also used to periodically re-initalize the Pagatisikos hydrodynamics model using variational initialization techniques. The ecosystem dynamics of Pagasitikos are tackled with a stand-alone implementation of the European Seas Ecosystem Model (ERSEM). The assimilation scheme is based on the Singular Evolutive Extended Kalman (SEEK) filter, in which the error statistics are parameterized by means of a suitable set of Empirical Orthogonal Functions (EOFs).The assimilation experiments were performed for year 2003 and additionally for a 9-month period over 2006 during which the physical model was forced with the POSEIDON-ETA 6-hour atmospheric fields. The assimilation system is validated by assessing the relevance of the system in fitting the data, the impact of the assimilation on non-observed biochemical processes and the overall quality of the forecasts. Assimilation of either GlobColour in 2003 or SeaWiFS in 2006 chlorophyll-a data enhances the identification of the ecological state of the Pagasitikos Gulf. Results, however, suggest that subsurface ecological observations are needed to improve the controllability of the ecosystem in the deep layers.