Bioactive compounds of the body wall of the sea cucumber (Holothuria leucospilota) collected from the Persian Gulf were extracted using methanol and analyze separation was determined by GC-MS. Extracts were evaluated for their antibacterial and antifungal activities against Gram positive and Gram negative bacteria and also Candida species by using disk diffusion and broth microdilution susceptibility tests. Efficacy of extract was evaluated against sessile yeast and bacterial cells by the time-dependent killing assay. GC-MS analysis clearly showed the presence of twenty important constituents. The major determined bioactive compound in methanolic extract was fatty acids. The preliminary test showed significant antimicrobial activity for all isolates tested (with zone of inhibition ranging from 7.1 ± 0.56 to 14.1 ± 0.91 mm). The inhibition zones for Candida species tested were ranged from 7.4 ± 0.75 to 14.7 ± 0.46 mm. The MICs value were ranged from 5 ± 0.02 to 80 ± 0.75 mg/ml for bacteria tested, while Candida species were more susceptible to extract and MICs were ranged from 0.75 ± 0.01 to 25 ± 0.90 mg/ml. All isolates tested were showed significant reduction of cells started from 2-6 h incubation. It is also indicated that Candida albicans was more susceptible in terms of decreasing the number of cells after 24 h incubation time in compare to other species.