A survey was carried out to determine the prevalence of anisakid nematode larvae in European anchovy (Engraulis encrasicolus) fished off the Tyrrhenian coast of central Italy. From February through July 2012, 1,490 specimens of E. encrasicolus caught in three different fishing areas (off Civitavecchia, Anzio, and Gaeta in the northern, central, and southern Lazio region of Italy, respectively) were tested for the presence of anisakid larvae, both by visual microscopic inspection and enzymatic digestion. In each of the three fishing areas, each of two sampling times produced 250 fish (with the exception of one sampling time in Gaeta that produced 240 fish). Larvae of the family Anisakidae were detected with an overall estimated prevalence of 2.3%, and each positive fish harbored a single larva. No anisakid larvae were detected in fish caught off Gaeta. Fish with larvae were significantly longer (standard length) than fish without larvae. Twenty-six larvae (74.3%) were detected by visual inspection of the viscera, eight larvae (22.8%) were detected by visual inspection of the fillets, and one larva (2.8%) was detected after digestion of pooled fillets. Molecular analysis to fully characterize the 35 detected larvae revealed 15 specimens of Anisakis pegreffii, 10 specimens of Hysterothylacium aduncum, and one hybrid genotype of A. pegreffii × Anisakis simplex. For nine specimens, no visible product was obtained after PCR amplification. The overall prevalence for A. pegreffii and H. aduncum was 1.0 and 0.7%, respectively. A comparison between fishes harboring A. pegreffii larvae and those harboring H. aduncum revealed that those with A. pegreffii were significantly heavier. The prevalence of anisakid larvae found in the present study is lower then that reported previously in E. encrasicolus collected in the Mediterranean Sea.