St. John's wort is a medicinal plant with a long history of use in traditional medicine all over Europe. Traditional preparations and in particular the infused oil from SJW flowers remains one of the most popular and curative topical remedy against ulcerations and burns. The presence of the characteristic polyprenylated acylphloroglucinol derivatives, namely hyperforin and analogs are instead related to the oil's therapeutic activity. Indeed, it is well known that hyperforin has a potent antibacterial activity. In this study we tried to rationalize the production system of the oily preparation in order to obtain the highest concentration and stability of phloroglucinols. Five different samples of SJW oils were evaluated by HPLC-DAD-MS analysis to verify the variability and stability of the constituents according to the following factors: different harvesting time, fresh or dried plant material, use of sunlight or heating systems during extraction. The stability of these oils during 1 year was also tested.