In recent years, actinobacteria have emerged as promising candidates for anti-cancer therapies, garnering significant attention in biomedical research. This study focuses on isolating and characterizing actinobacteria from diverse cave systems within Egypt's Beni-Suef governorate. The aim of the current study was to assess the biological activity, particularly antibacterial and anticancer properties, of the bioactive compounds produced by these actinobacteria. Through selective media and techniques, 112 strains were isolated from cave soil samples, subsequently purifying them for further analysis. Evaluation of secondary metabolites extracted from representative strains revealed significant antimicrobial activity against various pathogenic bacteria, yeast, and fungi, with a notable preference for Gram-positive bacteria. Additionally, five actinobacteria extracts have displayed potential antibacterial properties, while extracts from two strains, Am 139 and Am 152, exhibited substantial cytotoxic effects on multiple cancer cell lines. Further experiments confirmed their promising anticancer potential against MCF7, HepG2, and JURKAT cell lines across various concentrations. Our findings underscore the untapped potential of actinobacteria-derived metabolites from Beni-Suef caves. Moreover, the isolation of actinobacteria from previously unexplored sites such as caves suggests a rich source of bioactive compounds with antimicrobial and anticancer activities, promising new avenues for drug discovery and development.