Hypericum perforatum L., also known as St. John’s Wort, is recognized worldwide as a valuable medicinal herb; however, other Hypericum species were intensively studied for their bioactive potential. To fill the research gap that exists in the scientific literature, a comparative evaluation between H. alpigenum Kit., H. perforatum L. and H. rochelii Griseb. & Schenk was conducted in the present study. Two types of herbal preparations obtained from the aerial parts of these species were analyzed: extracts obtained through maceration and extracts obtained through magnetic-stirring-assisted extraction. LC-DAD-ESI-MSn analysis revealed the presence of phenolic acids, flavan-3-ols and flavonoid derivatives as the main constituents of the above-mentioned species. Moreover, all extracts were tested for their antioxidant, enzyme-inhibitory and antimicrobial potential. Our work emphasizes for the first time a detailed description of H. rochelii phenolic fractions, including their phytochemical and bioactive characterization. In comparison with the other two studied species, H. rochelii was found as a rich source of phenolic acids and myricetin derivatives, showing important antioxidant, anticholinesterase and antibacterial activity. The study offers new perspectives regarding the chemical and bioactive profile of the less-studied species H. alpigenum and H. rochelii.