A series of novel 1,3-diallyltrisulfane analogues were synthesized and assayed in vitro for antimicrobial activity against Gram positive, Gram negative bacteria and fungi. The antimicrobial activity of the 1,3-diallyltrisulfane derivatives showed, on the whole, very potent towards all the tested Gram positive, Gram negative and fungi (MIC ranging from 4 to 256 μg/mL). 1,3-Di(pent-4-enyl)trisulfane 3b and 1,3-bis(3-methylbut-2-enyl)trisulfane 3e exhibited the strongest antibacterial activity among all the compounds, and both of them were more active than 1,3-diallyltrisulfane (DATS). Results indicated the relationship of either carbon number or lipophilicity with antimicrobial activity presented “V” shape. These observations provided some predictions in order to further design 1,3-diallyltrisulfane derivatives with antimicrobial activity.