Phytochemical investigation on a crude methanolic bark extract of Goniothalamus velutinus resulted in the isolation of four compounds, aristolactam I (1), aristolactam BII (2), aristolactam AII (3) and velutinam (4). Compounds (1) and (2) have previously been isolated from the bark of G. velutinus but this is the first report on isolation of compounds (3) and (4) from this specie. Antibacterial activities of these compounds were tested against Gram-positive bacteria (Bacillus subtilis, Bacillus spizizenii and Staphylococcus aureus) but none of them showed any antibacterial activity under the tested concentrations. These compounds were also tested for anti-biofilm activity against Klebsiella pneumoniae, S. aureus, Streptococcus mutans and Proteus mirabilis and among the four compounds only velutinam (4) (70 µg/mL) inhibited biofilm formation of both S. mutans and P. mirabilis up to 41.3 and 78.9%, respectively. Whereas, the compounds aristolactam BII (2) (90 µg/mL) and aristolactam AII (3) (140 µg/mL) were active against S. mutans and inhibited their biofilm formation to 72.8% and 89.9%, correspondingly. In addition aristolactam I (1) did not show any inhibition of biofilm against the microorganism used in this study. The anticancer activity of velutinam (4) was evaluated against various cell lines including Human lung adenocarcinoma epithelial cells (A549), human embryonic kidney cells (HEK 293) and epidermoid cervical carcinoma cells (CaSki). The MTT antiproliferative assay resulted in IC50 values of 21.57 µg/mL, 13.28 µg/mL and 10.97 µg/mL for A549, HEK 293 and CaSki, respectively. Although anticancer activity of velutinam (4) has been reported on HeLa and L1210 cell lines, it has never been reported for the cell lines under this study. To our knowledge, this is the first report on anticancer activity of velutinam (4) on these cell lines.