This research was conducted to explore the prospect of fatty acid derivatives as candidates for anticancer and antimicrobial drugs. Synthesis of fatty alkanolamides was carried out by direct amidation of fatty acids with alkanolamines. Several fatty acids and alkanolamines were used as starting materials to investigate the effect of the chain length, the presence of C=C bond, and the number of hydroxyl group of alkanolamides on the cytotoxic activity against HeLa cell line and also antimicrobial activity against Escherichia coli and Staphylococcus aureus. The structure of synthesized alkanolamides was characterized using FTIR and 1H-NMR spectroscopy, the cytotoxic activity was evaluated using a standard MTT assay, and the antimicrobial activity was determined using the disc diffusion method. The HeLa cell line was cultured in DMEM medium supplemented with fetal bovine serum (FBS), penicillin, and streptomycin for 48 h during the cytotoxic assay. Chloramphenicol was used as positive control dan DMSO as a negative control for antimicrobial activity. Antimicrobial activity was conducted at 37 °C for 24 h. All fatty alkanolamines have been successfully synthesized in this research. All compounds demonstrated cytotoxic and antimicrobial activity as indicated and displayed accurately. A long-saturated alkyl chain was advantageous for the activities. Alkanolamide stearate-monoethanolamine showed the highest cytotoxic activity (IC50 = 37.5 μM) and antimicrobial activity. The presence of C=C bond decreased the activities. However, the polyunsaturated alkyl chain expressed greater activities than the monounsaturated alkyl chain. The number of hydroxyl groups on alkanolamines residue indicated a small contribution to the activity. Therefore, fatty alkanolamide is promising to be developed as an anti-cancer and antimicrobial drug. ©2024 National Information and Documentation Center (NIDOC)