Strawberries (Fragaria ananassa L., cv. favette) were studied to investigate the influence of cultivation practices (biodynamic, conventional) on the synthesis of bioactive molecules (ascorbic acid, ellagic acid, anthocyanins, flavonols) and to evaluate their antioxidant activity. Additionally, the in vitro bioactivity, in terms of antioxidant and antiproliferative activity, of the same strawberry samples in human colon carcinoma (Caco-2) cells was also studied. Compared to conventional strawberries, biodynamic fruits had a significantly higher content of ascorbic acid (P < 0.01), pelargonidin-3-glucoside (P < 0.05), cyanidin-3-glucoside (P < 0.01), ellagic acid (P < 0.01), quercetin, and kaempferol (both P < 0.01). Antioxidant activity of biodynamic strawberry crude extract was significantly higher than that of the conventional one (P < 0.05); in addition, while the antioxidant activity of water-soluble fraction was very similar in both biodynamic and conventional strawberries, that of water-insoluble fraction of biodynamic fruits was significantly higher (P < 0.05). The same crude extract of biodynamic strawberry samples effectively corresponded to an increase of bioactivity, in terms of both cellular antioxidant activity and antiproliferative activity, in Caco-2 cells differentiated to normal intestinal epithelia and in undifferentiated Caco-2, respectively. Further studies are needed to confirm whether the practice of biodynamic agriculture is likely to increase the bioactivity of other varieties of fruits and vegetables.