In this study, the effects of pure olive phenolic compounds and olive mill wastewater (OMWW) (after membrane filtration treatments) on Aspergillus flavus growth and aflatoxin B₁ (AFB₁) production, were investigated. Five OMWWs coming from Greek (Lianolia, Koroneiki and Asprolia) and Italian (Cellina di Nardò and Coratina) olive oil cultivars, opportunely filtered using a membrane system, were added at two concentrations (5 and 15%) to growth medium, in order to evaluate their effect on A. flavus growth and AFB₁ production. The OMWW fractions treatment, after 6 days of incubation, did not inhibit the fungal growth rate, but at 15% concentration significantly reduced the AFB₁ production (ranging from 88 to 100%). A similar approach was used for caffeic acid, hydroxytyrosol, tyrosol and verbascoside, the major pure phenolic compounds identified in OMWW fractions. They were evaluated at increasing doses (10, 50 and 100 µg/ml) following both AFB₁ production and fungal growth. At the highest concentration (100 µg/ml) all pure compounds showed a reduction of about 99% of AFB₁ production without any influence on fungal growth. This is the first time in which OMWWs and their main phenolics were used in the treatments against AFB₁ production. The results obtained could provide possible new strategies for preventing AFB₁ food contamination using olive polyphenols and OMWW fractions with anti-aflatoxigenic effect, and permitting to harness in a sustainable way an olive oil by-product.