Objectives Isolation, structure determination, and antimicrobial activities evaluation of compounds from the methanol extract of the marine microalga Thraustochytrium pachydermum TSL10. Methods Using chromatographic methods to isolate compounds from the methanol extract of the T pachydermum. The chemical structures were elucidated by analyses of HR-ESI-MS and NMR spectral data. The antimicrobial activities of compounds were evaluated using the dilution turbidimetric broth method as the standard protocol published by the Clinical and Laboratory Standard Institute. Results One new sterol, (24 R)-ergosta-7,22-diene-3 β,5 α,9 α-triol (1), and 16 known compounds, ergosta-7,22-diene-3 β,5 α-diol (2), cerevisterol (3), (22 E,24 S)-5 α,6 α-epoxy-ergosta-8(9),22-diene-3 β,7 α-diol (4), ergosterol (5), 5 α,8 α-epidioxyergosta-6,22-diene-3 β-ol (6), linoleic acid (7), 4-((9 Z,12 Z)-octadeca-9,12-dienoyloxy)butanoic acid (8), 1-linoleoyl-sn-glycero-3-phosphocholine (9), cibotiglycerol (10), gingerglycolipid B (11), oleic acid (12), 4-((9Z)-octadec-9-enoyloxy)butanoic acid (13), (2 S)-1- O-(9 Z)-octadec-9-enoyl)-3- O-(6-sulfo-D-quinovopyranosyl)glycerol (14), 6′- O-linoleylsucrose (15), gingerglycolipid C (16), and ( S)-3-stearoyl- sn-glycerol (17) were identified from the marine microalga Thraustochytrium pachydermum TSL10. Sterols 1–6 were found to exhibit significant antimicrobial activities on Gram-( + ) bacteria ( Enterococcus faecalis ATCC29212 , Staphylococcus aureus ATCC25923, and Bacillus cereus ATCC14579) and the yeast ( Candida albicans ATCC10231) with MIC values ranging from 32 to 256 µg/mL. Conclusions The results suggested sterols 1–6 could be potential antimicrobial agents against Gram-( + ) bacteria.