AbstractDNase I inhibitory potential of water extract of nine Hypericum species (H. umbellatum, H. barbatum, H. rumeliacum, H. rochelii, H. perforatum, H. tetrapterum, H. olympicum, H. hirsutum, H. linarioides) and the most important Hypericum secondary metabolites (hypericin, hyperforin, quercetin, and rutin) was investigated. All examined Hypericum extracts inhibited DNase I with IC50 below 800 μg/ml, whereby H. perforatum was the most potent (IC50=391.26±68.40 μg/ml). Among the investigated Hypericum secondary metabolites, rutin inhibited bovine pancreatic DNase I in a non‐competitive manner with IC50 value of 108.90±9.73 μm. DNase I inhibitory ability of rutin was further confirmed on DNase I in rat liver homogenate (IC50=137.17±16.65 μm). Due to the involvement of DNase I in apoptotic processes the results of this study indicate the importance of frequent rutin and H. perforatum consumption in daily human nutrition. Rutin is a dietary component that can contribute to male infertility prevention by showing dual mechanism of sperm DNA protection, DNase I inhibition and antioxidant activity.