A series of 3-polyaminochenodeoxycholic acid derivatives were synthesized and their in vitro antimicrobial and antifungal activities were assessed. The antimicrobial activity was evaluated against Gram-positive and Gram-negative bacteria, and the antifungal activity was assessed against two strains Candida albicans (ATCC MYA-1003) and Aspergillus fumigatus (ATCC 16424). The introduction of an amine group to steroid was accomplished by reductive amination of 3-oxosteroid 9 with Boc-spermidine and Boc-spermine, in the presence of NaBH(OAc) 3 . This afforded a high yield of 3-polyaminosteroids of 10–13. The 3β-sperminyl-5β-cholane 6 showed the highest antimicrobial activity against Streptococcus pyogenes 308A, Staphylococcus aureus 503, Escherichia coli DC2 and Pseudomonas aeruginosa 9027 with a MIC value of 3.13μg/mL.