This study investigated the phytochemical composition of Gossypium barbadense stem extracts and evaluated their antimicrobial and cytotoxic activities. Headspace GC/MS analysis identified 48 volatile compounds, including 13 major constituents representing over 56% of the total composition, while LC-ESI-MS identified 16 major phenolic and flavonoid compounds in the methanolic extract for the first time. The methanolic extract exhibited stronger antibacterial activity than the petroleum ether extract, particularly against Staphylococcus aureus and Escherichia coli, whereas neither extract showed antifungal activity. In cytotoxicity assays, the methanolic extract demonstrated potent activity against HEPG2 liver cancer cells and moderate activity against colon (HCT116) and breast (MCF7) cancer cell lines. The study concludes that G. barbadense stems are a valuable source of bioactive compounds with promising antimicrobial and anticancer potential.