The ability of endophytic fungi to produce valuable bioactive compounds when surviving in medicinal herbs. Finding out endophytic fungi originated from Catharanthus roseus with antimicrobial, antioxidant and cytotoxicity activities is important for pharmaceutical development. The isolation was based on the morphology of fungi. Identification was performed by sequencing 18S rRNA that was compared with known genes using Blast search in the combination of phylogenic analysis by using Clustal W and PhyML in GenomeNet. For the antimicrobial test, the agar diffusion method was used. DPPH scavenging assay for the antioxidant activity determination by using spectrophotometry. The cytotoxicity test was carried out by Sulforhodamine B method. LC-MS was applied for predicting components. We isolated the Fusarium oxysporum F01 strain originated from Catharanthus roseus. The aqueous extracts showed inhibition against Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa ATCC 27853 (15.00 ± 0.50 mm), Serratia marcescens ATCC 14756 (9.00 ± 0.87 mm), Vibrio parahaemolyticus ATCC 17802 (15.50 ± 0.87 mm), Escherichia coli ATCC 25922 (12.50 ± 0.50 mm). The antioxidant activity of the extract obtained from the supernatant was determined with an IC50 value of about 11 µg/mL. The extract also showed cytotoxicity effect on liver cancer cell line (HepG2) and breast cancer cell line (MCF-7) with the percentage of inhibition of 84.47 ± 3.18 and 94.69 ± 1.59, respectively. LC-MS was used to point out the presence of pratol, a melanogenesis agent. The study provided more interesting information about Fusarium oxysporum F01 isolated in Catharanthus roseus grown in Vietnam, contributing to pharmaceutical sources in the world.