Our research evaluated the antimicrobial and cytotoxic activities of crude extracts of endophytic fungi from R. stylosa and R. mucronate. 46 fungal isolates were cultured on the four different mediums, namely Dextrose Agar (PDA), Czapek’s Agar (CZA), Rice Medium (RM) and Grain Medium (GM), and harvested by ethyl acetate solvent at 40 days. The extracts were tested for antimicrobial activity by the microdilution method against Gram-negative bacteria Pseudomons adaceae (PA), Gram-positive bacteria Enterococcus faecalis (EF), Methicillin-resistant Staphylococcus aureus (MRSA) and pathogenic fungus Monilia albican (MA). Cytotoxic activity of the extracts was evaluated by MTT assay using human lung cancer cells A549, human cervical carcinoma cells Hela, and human hepatocellular cells HepG2. The results showed that rice medium could promote the secretion of antimicrobial and anti-tumor secondary metabolites of endophytic fungi in comparison with the other cultivation media. 17 strains (68%) from R. stylosa exhibited inhibition effects on indicators, especially N. protearum HHL46 which could inhibit the growth of four microbes with the MIC values reaching 0.0625 mg/mL. 15 strains (71.4%) from R. mucronata displayed activities against human pathogenic microbes, especially Pestalotiopsis sp. HQD6 and N. protearum HQD5 which could resist the growth of four microbial with MIC values ranging from 0.015 to 1 mg/mL. In cytotoxic assay, the extracts of 10 strains (40%), 9 strains (40%) and 13 strains (52%) from R. stylosa and 13 strains (61.9%), 10 stains (47.6%) and 10 stains (47.6%) in R. mucronata displayed the cytotoxicity against A549, Hela and HepG2 cancer cells with cells viability value ≤50%, respectively. Neopestalotiopsis protearum HHL46, Phomopsis longicolla HHL50, Botryosphaeria fusispora HQD83, Fusarium verticillioides HQD48 and Pestalotiopsis sp. HQD6 displayed significant antitumor activity with IC50 values below the 20 μg/mL. These results highlighted the antimicrobial and anti-tumor potential of endophytic fungi from R. stylosa and R. mucronata and the possibility to be exploited for their antimicrobial and cytotoxic agents.