The naturally occurring (5Z,9Z)-5,9-hexadecadienoic acid was synthesized stereochemically pure in six steps starting with commercially available 1,5-hexadiyne. The title compound was antimicrobial against the Gram-positive bacteria Staphylococcus aureus (MIC 80 microM) and Streptococcus faecalis (MIC 200 microM), but inactive against Gram-negative bacteria such as Pseudomonas aeruginosa. In addition, the (5Z,9Z)-5,9-hexadecadienoic acid completely inhibits human topoisomerase I at a concentration of 800 microM, while 5,9-hexadecadiynoic acid and hexadecanoic acid do not inhibit topoisomerase I (>1000 microM). This comparison reveals that the cis double bond geometry in the title compound is required for topoisomerase I inhibition. Moreover, these results suggest that the antimicrobial activity of (5Z,9Z)-5,9-hexadecadienoic acid against either S. aureus or S. faecalis could be a result, at least in part, of the inhibitory activity of the acid against topoisomerases.