The lack of deep knowledge of plant pathogenic fungal biofilms is reflected in the few existing environmental-friendly options for controlling fungal plant disease. Indeed, chemical fungicides still dominate the market but present-day concerns about their real efficacy, increasing awareness of the risk they pose to human health and the environment, and the incidence of fungicide resistance have all led to the current trend of near zero-market-tolerance for pesticide residues in fruit and vegetables. Here, essential oils (PK and PK-IK) from the edible leaves of two cultivars of Perilla frutescens are proposed as new, effective, non-toxic, eco-friendly pesticide-free options suitable for a preventive or integrative approach for sustainable crop protection and product preservation. PK and PK-IK were extracted and characterized, and their ability to affect the biofilm formation of the phytopathogenic model fungi Colletotrichum musae, Fusarium dimerum and Fusarium oxysporum was studied at non-lethal doses. Both essential oils at 1000 and 2000 mg l-1 showed excellent anti-biofilm performance: i) reducing conidia adhesion up to 80.3 ± 16.2%; ii) inhibiting conidia germination up to 100.0 ± 0.0%; iii) affecting biofilm structural development, with a reduction in dry weight of up to 100.0 ± 0.0% and extracellular polysaccharides and proteins up to 81.4 ± 8.0% and 51.0 ± 6.1% respectively. In all cases PK-IK showed better activity than PK.