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Molecular techniques are offering new insights on the feeding strategy and diets of planktonic organisms such as the larvae of marine fish. We applied multiplex PCR to provide the first estimates of the diets of the larvae of European sardine (Sardina pichardus) in the Alboran Sea (SW Mediterranean), where this species represents an important fishery resource. The feasibility of this technique was tested in a 26-hour continuous survey of a shoal of larvae (mean±SD, 10.80±0.73 mm in standard length). Multiplex PCR was designed to detect the presence of five copepod species, a microplanktonic dinoflagellate (Gymnodinium) and the picoeukaryote algae family Prasinophyceae in larval guts. We simultaneously sampled sardine larvae and their potential prey (pico-to mesoplankton) and compared diel variability of the prey field and ingested items.Microplankton was dominated by flagellates and copepods represented the most abundant mesozooplankton, reaching peak abundance at night. Prey DNA was detected throughout the entire diel cycle, despite no visible prey in the guts of larvae collected at night. Sardine larvae preyed on early life stages of the most abundant copepod species (Oncaea waldemari, Paracalanus indicus and Temora stylifera), suggesting an opportunistic foraging behavior. The use of multiplex PCR allowed species-level identification of ingested nauplii and protists, which otherwise would remain unidentified.