Hyaluronidase is an ubiquitious enzyme, present, amongst others, in hymenoptera venom and in medical formulations. The latter include use as an emergency treatment or to correct undesired outcomes of medical and aesthetic procedures using hyaluronic acid fillers. By performing detailed allergy work-ups including prick-testing we investigated here if patients with a history of allergic reaction to hymenoptera venom are also sensitized to medical grade hyaluronidase. Ninety patients with a history of type-1 reaction to hymenoptera venom with and without a history of previous specific venom immunotherapy were included in the study. All underwent skin prick tests for medical hyaluronidase. All patients also underwent serological analysis for Api m2, the only commercially available IgE-test for a hymenoptera hyaluronidase. Of the 90 patients with previous type-1 reactions to hymenoptera venom hyaluronidase included in the study, 60 had undergone previous venom immunotherapy, 30 did not. A majority (73/90) were allergic to wasps, followed by honeybees (14/90) and 3 were allergic to both. Neither patients having undergone previous immunotherapy nor those allergic to bees showed positive skin prick tests to medical hyaluronidase. Of those with a wasp allergy and naïve to immunotherapy, over 20% (5/23) showed positive skin prick tests to medical hyaluronidase. Healthy controls (0/30) without previous allergic reactions to hymenoptera did not show positive skin prick tests to medical hyaluronidase. Sensitization to hyaluronidase is most common in wasp-allergic patients who have not had previous specific immunotherapy. As allergic reactions to medical hyaluronidase are reported to be scarce, this group is probably at the highest risk to develop anaphlaxis to medical hyaluronidase. While all patients with untreated anaphylaxis to hymenoptera venom should consult an allergy specialist, particularly those with untreated wasp allergies need to seek a specialist's advice before treatment with medical hyaluronidase is initiated.