Phallusia mammillata (Cuvier, 1815) is a tunicate which has adapted to the harsh marine environment by production of the secondary bioactive metabolites that provide protection from predators, aid in hunting process and defend their territory against invading competitors. Because of their unique chemical structures and diverse biological activities, marine secondary metabolites can be used to design and develop new potentially useful therapeutic agents. Majority of studies using P. mammillata investigated its basic development processes, but biological activities of tunicate extracts or its secondary metabolites have not been thoroughly described. Thus, the evaluation of in vitro biological potential of crude organic extracts of tunicate P. mammillata was conducted. To determine the most suitable organic solvent for the extraction process, the organism was extracted by using five solvents of different polarity: petroleum ether, dichloromethane, ethanol, methanol and acetonitrile. Petroleum ether and ethanol were the most suitable nonpolar and polar solvents, respectively, based on extraction yields. Antioxidant activity of P. mammillata crude organic extracts was tested by ABTS and DPPH assays. Antiproliferative and cytotoxic activities, expressed as IC50 and LC50 values, respectively, were evaluated by MTT assay by using following tumor cell lines: colorectal carcinoma (HCT116), ductal pancreatic adenocarcinoma (CFPAC-1), metastatic breast cancer (MCF7), hepatocellular carcinoma (HepG2) as well as non-transformed skin fibroblasts (HFF). Alcoholic and acetonitrile extracts showed significant ABTS scavenging activity. Significant DPPH scavenging activity was not observed in any of the organic extracts. Only petroleum ether extract demonstrated significant antiproliferative and cytotoxic activity against tumor cell lines. To evaluate which natural products classes may be responsible for observed biological activities, the presence of bioactive metabolites in P. mammillata organic extracts was evaluated by specific and qualitative colorimetric tests. Qualitative chemical screening of organic extracts revealed the presence of phenolic compounds and alkaloids in each tested sample. Terpenoids, sterols and glycosides were present in petroleum ether and in acetonitrile extract, while absent in the rest of samples. Saponins were detected only in acetonitrile extract.