In the course of structure-activity relationships on rebeccamycin analogues, a series of compounds bearing a halogenoacetyl substituent were synthesized with the expectation of increasing the interaction with DNA, possibly via covalent reaction with the double helix. Two rebeccamycin analogues bearing an acetyl instead of a bromoacetyl substituent were prepared to gain an insight into the role of the halogen atom. The new compounds show very little effect on protein kinase C and no covalent reaction with DNA was detected. However, the drugs behave as typical topoisomerase I poisons, and they are significantly more toxic toward P388 leukemia cells than to P388/CPT5 cells resistant to camptothecin. The introduction of a bromo- or chloro-acetyl substituent does not affect the capacity of the drug to interfere with topoisomerase I either in vitro or in cells. One of the bromoacetyl derivatives, compound 8, is the most cytotoxic rebeccamycin derivative among the hundred of derivatives we have synthesized to date. In addition, we determined the antimicrobial activities against two Gram-positive bacteria, Bacillus cereus and Streptomyces chartreusis, and against the Gram-negative bacterium Escherichia coli. The effect of the drugs on Candida albicans yeast growth and their anti-HIV-1 activities were also measured.