This study determined the favourable fermentation conditions for the production of antifungal peptides from kenaf seeds and their effectiveness in extending the shelf-life of tomato puree. The optimum fermentation conditions for the maximum activity of the antifungal peptides were 8.4% (w/v), 7 days and 3.7% for substrate/water ratio, fermentation time and glucose concentration, respectively. Eight cationic peptides of low molecular weight ranging from 840 to 1876 Da were identified in kenaf seed peptides mixture (KSPM). The minimum inhibitory concentration and minimum fungicidal concentration of KSPM against Fusarium sp. were 0.18 mg/mL and 0.70 mg/mL, respectively, while those for Aspergillus niger were 1.41 mg/mL and 2.81 mg/mL respectively. KSPM exhibited a fungicidal effect and a prolonged lag phase, with increased fungal membrane permeability as the concentration of KSPM increased, as evidenced by the release of intracellular constituents. The treatment of tomato puree with 1000 mg/kg KSPM delayed fungal growth for up to 14 and 23 days at 25 °C and 4 °C respectively, significantly reducing Aspergillus niger and Fusarium sp. counts. In conclusion, kenaf seed peptides prepared by lacto-fermentation possess antifungal activity, hence can be applied as natural bio preservatives to extend the shelf-life of food products such as tomato puree.