Three new compounds; beta-sitosterol-3-O-(3Z)-pentacosenoate (1), 5 alpha-pregna-3 beta-acetoxy-12 beta, 16 beta-diol-20-one (2), and echinoclathriamide ((R)-2'-hydroxy-N-((2S, 3S, 4R)-1,3,4-trihydroxy-19-methylicosan-2-yl) heptadecanamide) (3), together with two known compounds; thymine (4) and uracil (5) were isolated from the EtOAc fraction of the Red Sea sponge Echinoclathria gibbosa. Their structures were unambiguously established on the basis of 1D and 2D NMR spectroscopy, in addition to mass spectrometry. The total MeOH extract (TME) and its fractions were evaluated for their antimicrobial, anti-inflammatory, antipyretic, and hepato-protective activities. The in vitro growth inhibitory activity of the isolated compounds was evaluated against three human cancer cell lines including the A549 non-small cell lung cancer (NSCLC), U373 glioblastoma (GBM), and PC-3 prostate cancer cell lines. Compound 1 showed weak activity against the three cancer cell lines. (C) 2014 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.