Monoglucosyl diglycerides with medium-long length fatty acid acyl chains were prepared and examined for antimicrobial activity against Gram-positive, Gram-negative bacteria and fungi. The study of their in vitro antimicrobial activity confirms the significant activity of some monoglucosyl diacylglycerol analogues and establishes for the glucose series that the 1,2-disubstitution and the octanoyl chain are the proper structural features for the maximum activity.