Three distinct fractions containing polar glycolipids (PF(1-3)) were isolated from the chloroform soluble fraction of crude methanolic extract of red alga Chondria armata (Kütz.) Okamura on gel chromatography over Sephadex LH20. Their structure was elucidated by multidimensional nuclear magnetic resonance (NMR) techniques like 1H, 1H correlation spectroscopy (COSY), 1H, 1H total COSY (TOCSY), 1H, 13C heteronuclear multiple quantum coherence (HMQC), and 1H, 13C heteronuclear multiple bond correlation (HMBC) complemented by electrospray ionization mass spectrometry (ESI-MS) in the positive ion mode. The coupling constant of the anomeric proton in 1H NMR spectrum and sign of rotation indicated an exclusive configuration of the sugar molecules in the glycerolipids. Major glycolipids were identified as (2R)-2-O-(5,8,11,14-eicosatetranoyl)-3-O-alpha-d-galactopyranosyl-sn-glycerol (GL2), its pentacetate (GL1), and (2R)-1-O-(palmitoyl)-2-O-(5,8,11, 14,17-eicosapentanoyl)-3-O-beta-d-galactopyranosyl-sn-glycerol (GL3). Each was methanolysed to give the same galactosylglycerol which on ESI-MS provided a pseudomolecular ion at m/z 309 representing deacylated glycolipid with the sodiated sugar moiety. Additionally, six minor glycolipids were also identified on the basis of ESI-MS. These include a 1,2-di-O-acyl-3-O-(acyl-6'-galactosyl)-glycerol (GL1a), sulfonoglycolipids 2-O-palmitoyl-3-O-(6'-sulfoquinovopyranosyl)-glycerol (GL2a) and its ethyl ether derivative (GL2b), 1-oleoyl-2-palmitoyl-3-O-galactosyl glycerol (GL3a), and 1,2-diacyl phosphatidyl glycerol (GL3b). GL1, GL1a, and GL2b are new to the literature. The novelty of the remaining identified compounds lies in the diversity of their fatty acid composition. Antimicrobial properties of these glycolipids against pathogens were evaluated. The yeast Candida albicans and the bacteria Klebsiella sp. were as sensitive as the standard Nystatin and antibiotic Streptomycin against PF3. Considerable activity was expressed by the same metabolite against the fungus Cryptococcus neoformans as compared to the control. Weak activity against the bacteria Shigella flexineri and Vibrio cholerae and the fungus Aspergillus fumigatus was also observed. Fraction PF2 was weakly active against some strains whereas all of them were resistant to its acetyl derivative PF1. Antimicrobial activity of glycolipids is being reported here for the first time.