The quorum sensing inhibitory (QSI) and antimicrobial potentials of the total methanol extract (TME) and different extractives as well as the sesquiterpenes: dehydrodihydrocostus lactone (1), dehydrocostus lactone (2), arbusculin A (3), santamarine (4), reynosin (5), and specioic acid (6) isolated from Costus speciosus rhizomes were evaluated. The CHCl3:EtOAc (1:1), EtOAc, and TME fractions exhibited potent antibacterial activity toward B. cereus with inhibition zone diameter 13 mm. While the CHCl3 fraction showed strong activity towards S. aureus and B. cereus with inhibition zone diameter 11 and 19 mm, respectively. Moreover, the TME and CHCl3 fractions have strong activity towards C. albicans with inhibition zone diameter 15 and 12 mm, respectively. Compound 5 showed prominent activity towards B. cereus (MIC 385 μg/mL). However, 6 exhibited significant activity with MIC values of 150, 400, and 550 μg/mL towards S. aureus, E. coli, and B. cereus, respectively. Moreover, it showed potent antifungal effect towards C. albicans (MIC 320 μg/mL). Most of the tested fractions had QSI activity against C. violaceum. Only compound 6 exhibited moderate QSI effect with disappearance of violet pigment. In addition, compounds 1-6 were evaluated for their in vitro antiproliferative activity towards KB, BT-549, SK-MEL, and SKOV-3 cancer cell lines.