Ferns are vascular plants with significant ornamental value, and in recent years, they have also been considered for the potential application in different economic sectors like nutrition, medicine and cosmetics. This study aimed to obtain in vitro fern gametophytes and sporophytes and evaluate their potential as secondary metabolites sources. For this study four fern species were used: Polypodium vulgare L. (PV), Asplenium trichomanes L. (AT), Athyrium filix-femina L. Roth (AF), and Osmunda regalis L. (OR). The polyphenols, flavonoids, tannins and terpenoids content and antioxidant activity were estimated by different colorimetric methods. For metabolite identification and their antioxidant activity, HP-TLC separations were used. Also, quantitative HPLC–DAD analysis was performed to estimate the content of certain metabolites. The results showed that in vitro generation of gametophytes registered higher growth rates in OR, PV, and AF, while the regeneration of sporophytes was better for PV, AT, and AF. The OR gametophyte produced the highest quantities in all analyzed metabolite classes and also had the highest antioxidant values. We report for the first time the presence of metabolites such as catechin, caffeic acid, gallic acid, ferulic acid, cinnamic acid, or rutin in OR. In this study, we demonstrated the possibility of producing potent antioxidant metabolites by in vitro cultures in ferns.