Research of antimicrobial activity and cytotoxic activity from isolates symbiotic fungi from marine sponge Xestospongia testudinaria has be done. Xestopongia testudinaria collected from Mande, South Pesisir, West Sumatera. Isolation symbiotic fungi using sabouraud dextrose agar (SDA) as growth medium. Seven isolates symbiotic fungi strains were obtained from this sponge Xestospongia testudinaria. Symbiotic fungi isolate was cultivated using rice medium for 40 day. The cultivation yield was extracted using an ethyl acetate, from the extraction result obtained the weight ethyl acetate extract which varied that 320-756 mg.The ethyl acetate extracts were analyzed for antimicrobial and cytotoxic activities by using diffusion agar method and BSLT. The research revealed 71.42% of the total extract had antimicrobial activity against pathogenic bacteria and fungal such as, Staphylococcus aureus, Pseudmonas aeruginosa and Candida albicans and 71% were cytotoxic activity with Artemia salina L. This selected fungal isolates were first macroscopically and microscopically characterized and later molecularly identified as Penicillium citrinum. This research concluded that the sponge Xestospongia testudinaria symbiotic fungi can be developed as a new source of antibiotic and anticancer compounds.