Serodiagnosis of human anisakidosis is presently hampered by the current lack of standardised serological assays that allow sensitive and specific detection of Anisakidae-specific antibodies in human patients. In the present study, we comparatively evaluated the diagnostic value (by IgG-ELISA) of excretory-secretory antigens (ESAgs) of Anisakis simplex, Pseudoterranova decipiens and Contracaecum osculatum, representing the most frequently found genera responsible for human infection. In addition, we tested also a mix of the three ES preparations (Mix-ESAgs) as well as two recombinant allergens of A. simplex, rAni s 1 and rAni s 7. ES antigen from C. osculatum yielded the best diagnostic performance in IgG-ELISA-based serodiagnosis of the Spanish anisakidosis patients investigated in this study (relative serodiagnostic sensitivity 100%; specificity 89%) as compared to A. simplex ES-antigen (93% versus 57%) and P. decipiens (67% versus 93%) or a mix of the three ES antigens (100% versus 44%), respectively. Cross-reactions of C. osculatum ES antigen with serum-antibodies from patients suffering from other helminth infections were rare and were exclusively found with few sera from toxocariasis, ascariasis, and filariasis patients. The two recombinant allergens rAni s 1 and rAni s 7 did not prove sufficiently sensitive and specific in order to justify a further evaluation of these antigens regarding their suitability in IgG-ELISA-based serodiagnosis of human anisakidosis. In conclusion, the C. osculatum-ESAg-ELISA remains as key candidate to be further assessed for the serodiagnosis of symptomatic anisakidosis in different endemic regions.