Starting from 3 beta-acetoxy-17a-homo-17-oxa-androst-5-en-16-one (1) and in the reaction with N-bromoacetamide (NBA) and perchloric acid, the 5 alpha-bromo-6 beta-hydroxy (2), 5 beta,6 beta-epoxy (3) and 5 alpha,6 beta-dibromo (4) derivatives were obtained. The structure of compounds 2-4, particularly stereochemistry at C5 and C6, is established by detailed NMR and X-ray analysis. The in vitro antiproliferative activity of newly synthesized compounds 2-4 against six human tumor cell lines was evaluated. All three compounds showed a significant toxicity toward hormone-independent breast adenocarcinoma MDA-MB-231 and cervical carcinoma cells HeLa, while dibromo derivative 4 was active toward five human cancer cell lines. These new 5,6-disubstituted-D-homolactone steroidal compounds have also displayed selectivity toward cancerous cell lines against inactivity found for noncancerous control cell line. This selectivity was not found for control compound, well-known chemotherapy drug cisplatin.