The growth rates of planktonic microbes in the pelagic zone of the Eastern Mediterranean Sea are nutrient limited, but the type of limitation is still uncertain. During this study, we investigated the occurrence of N and P limitation among different groups of the prokaryotic and eukaryotic (pico-, nano-, and micro-) plankton using a microcosm experiment during stratified water column conditions in the Cretan Sea (Eastern Mediterranean). Microcosms were enriched with N and P (either solely or simultaneously), and the PO4 turnover time, prokaryotic heterotrophic activity, primary production, and the abundance of the different microbial components were measured. Flow cytometric and molecular fingerprint analyses showed that different heterotrophic prokaryotic groups were limited by different nutrients; total heterotrophic prokaryotic growth was limited by P, but only when both N and P were added, changes in community structure and cell size were detected. Phytoplankton were N and P co-limited, with autotrophic pico-eukaryotes being the exception as they increased even when only P was added after a 2-day time lag. The populations of Synechococcus and Prochlorococcus were highly competitive with each other; Prochlorococcus abundance increased during the first 2 days of P addition but kept increasing only when both N and P were added, whereas Synechococcus exhibited higher pigment content and increased in abundance 3 days after simultaneous N and P additions. Dinoflagellates also showed opportunistic behavior at simultaneous N and P additions, in contrast to diatoms and coccolithophores, which diminished in all incubations. High DNA content viruses, selective grazing, and the exhaustion of N sources probably controlled the populations of diatoms and coccolithophores.