Musa balbisiana Colla is extensively found in South-East Asia. Its fruit is widely consumed around the world and it is rich in natural products. The natural products found in the fruit pulp of Musa balbisiana Colla can be a better potential candidate for future alternative medicinal drugs to be designed or synthesized. Hence, evaluating and studying the acute, subacute toxicity of Musa balbisiana Colla fruit pulp is very important. Swiss albino mice were treated orally with four different doses of methanolic extract of Musa balbisiana Colla fruit pulp and after administration they were screened for toxicity for two weeks. Different doses of the extract were given to multiple test groups of mice for 28 days throughout subacute toxicity study. During this study, overall changes of the body and major organ weight, hematological, serum biochemical and histopathological parameters were observed. The study indicated that the LD50 of the methanolic extract of Musa balbisiana Colla fruit pulp was exceeding 5000 mg/kg. Subacute toxicity study showed no significant negative impact on food intake, body and organ weights, hematological and serum biochemical parameters, mortality and histopathology of mice of different treatment groups.