In the present study, sea urchin; Diadema setosumwas collected from the Egyptian Red Sea coastline for investigating its antimicrobial activities. The physicochemical parameters of seawater samples were evaluated at sites where D. setosum was sampled. The data exhibited a little bit of variation of hydrographical measurements at the collection sites. In addition, the concentrations of dissolved nutrients in all stations were low, which revealed the oligotrophic condition of the Red Sea. The crude extract of D. setosum, was evaluated for antimicrobial activity against 15 pathogen isolates of bacteria, yeast, and fungi. The results showed fluctuations in antimicrobial activity values. The pathogens (Enterococcus faecalis ATCC 29219, Staphylococcus epidermidis ATCC 12228, Klebsiella pneumoniae ATCC13883, Bacillus subtilis ATCC 6633, Vibrio fluvialis, and Candida albicans ATCC10237) were not affected, while the other pathogens were clearly influenced. The positive values were recorded in the range of 9.3 to 18.0 mm. Additionally, the minimum inhibitory concentration (MICs) obtained from the crude extract of D. setosum were in the range of; 25 to 50 µg/mL against the affected microbes. Moreover, the activity of several commercial antibiotics was examined and compared with the results of D. setosum crude extract. Gram-positive bacteria showed obvious susceptibility towards most of the tested antibiotics, while Gram-negative ones showed more resistance. It was observed that the inhibition of D. setosum crude extract was lower than the potent commercial antibiotics in many cases. On the other side, the results of GC-MS/MS analysis of the crude extract revealed the presence of several bioactive constituents. Actually, it had 18 major compounds most of them known to possess antimicrobial activities.