The potential of marine crabs as a source of biologically active products is largely unexplored. In the present study, antimicrobial activity of the hemolymph (plasma) and hemocytes (plasma cells) of six brachyuran crabs was investigated against 16 pathogenic strains. Among the 16 strains tested maximum zone of inhibition was recorded in the hemolymph of Hyas araneus against Shigella flexineri. Interestingly Staphylococcus aureus and Salmonella typhi were susceptible to all the hemolymph and hemocytes samples. Likewise, the highest zone of inhibition was exhibited by both hemolymph and hemocytes samples against Vibrio cholerae. On the basis of TLC, 1HNMR, and 13CNMR it may be concluded that the antimicrobial activity in the hemolymph extract is due to the presence of lipids. This observation is further supported by the ESI-MS of the methanolic extract of hemolymph of H. araneus. ESI-MS shows cluster of peaks in the region m/z 445 to m/z 491 due to lysoglycerolipids/glycerides and cluster of signals between m/z 216 and 246, due to fatty acids/esters present in the sample.