Secondary metabolites constitute an extremely diverse class of compounds produced by plants, fungi, lichens, and microorganisms. Although they do not directly participate in basic life processes, they perform key ecological and adaptive functions: they facilitate chemical communication, defense against pathogens and herbivores, tolerance to environmental stress, and allelopathy. From the human perspective, many of them exhibit valuable biological activity (antibacterial, antifungal, antiviral, anti-inflammatory, antioxidant, and cytotoxic), which for decades has made natural products one of the most important sources of inspiration for pharmacotherapy and food technology. Synthetic reviews confirm that nature consistently provides new leading structures (leads) and drugs, ranging from antibiotics and anticancer agents to modern antibody–drug conjugates and therapies for lifestyle diseases [1,2,3]. From a scientific point of view, research on “Secondary Metabolites from Natural Products: Extraction, Isolation and Biological Activities” has several complementary goals. First, it allows for the scientific substantiation of traditional and ethnopharmacological uses of plants—from verifying historical indications to identifying the compounds responsible for the observed effect. Second, it enables the “rediscovery” of species described in historical sources and the exploration of biodiversity in new ecosystems (equatorial forests, Arctic and desert extremes, the deep sea, etc.). Third, it supports the search for alternative drugs, nutraceuticals, and functional ingredients through the integration of phytochemistry, microbiology, and molecular biology with modern analytics (LC–HRMS/MS, metabolomics, etc.). Finally, it offers pathways to sustainable applications in agriculture (biopesticides, allelochemicals), the food industry (natural preservatives, antioxidants, colorants, and emulsifiers), and even in industrial chemistry (biocatalysts, “green” reaction media, etc.). These lines of research are consistent with the European “Farm to Fork” strategy within the European Green Deal, which assumes, among other things, a 50% reduction in the use of and subsequent risk posed by chemical pesticides by 2030 [4,5,6,7,8,9]. In this Special Issue, we present cross-sectional examples of the implementation of these assumptions: from the optimization of extraction and comparison of methods for polyphenol-rich plant materials (e.g., Scutellaria baicalensis), through the use of supercritical techniques to obtain fractions with biological activity (e.g., Sorbus aucuparia), to the isolation and characterization of new structures from plants and fungi (meroterpenoids from Garcinia caudiculata, secobutanolides from Lindera obtusiloba, and metabolites of Dentipellis fragilis and endophytic Xylaria). Complementing these are review works (e.g., Calendula officinalis and Foeniculum vulgare, penicillides of Penicillium/Talaromyces, etc.), which organize knowledge and indicate research gaps, as well as studies in the area of ecophysiology and safety (allelopathy of lichens; Maillard reaction products as antioxidants). Together, they outline a map of current opportunities, from methodology and dereplication to applications in human health and agroecology.