Chalcone–isoxazole hybrids have emerged as a promising class of bioactive molecules with diverse pharmacological applications, particularly in antimicrobial and anticancer therapy. The structural fusion of the chalcone framework with an isoxazole moiety enhances biological potency and broadens therapeutic potential. Several derivatives (compounds 25–28) demonstrated potent anti-tuberculosis activity against Mycobacterium tuberculosis with low cytotoxicity, with compound 27 identified as a lead candidate. Structure–activity relationship (SAR) studies highlighted the role of nonpolar substituents, such as halogens and alkyl groups, in improving antimicrobial efficacy. Similarly, incorporation of thiazole–isoxazole motifs significantly strengthened antiproliferative activity. Notably, compound 38 exhibited outstanding cytotoxicity across multiple cancer cell lines, including MCF-7, A549, Colo-205, and A2780, with submicromolar IC₅₀ values. Although most derivatives displayed moderate to good cytotoxicity, their multifunctional properties—including antibacterial, antioxidant, and antiproliferative effects—underscore their versatility as scaffolds for drug discovery. Future efforts focusing on substituent optimization and hybrid design strategies may lead to the development of broad-spectrum therapeutic agents with enhanced efficacy.