Fourteen fungal endophytes were isolated from the Ocimum basilicum var. thyrsiflora leaves collected from Northern Thailand. Eight genera were identified including Aspergillus, Ascochyta, Nigrospora, Blastomyces, Colletotrichum, Exidia, Clitopilus, and Nomuraea. The antibacterial activity of crude extracts from all endophytic fungi was tested against nine human bacterial pathogens: Staphylococcus aureus, Staphylococcus epidermidis, Bacillus subtilis, Bacillus cereus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Vibrio cholerae, and Vibrio parahaemolyticus. All crude extracts showed some degree of antibacterial activity, but the crude extract from Nigrospora MFLUCC16-0605 exhibited broad spectrum activity with MIC values ranging from 7.81 to 250 A mu g/mL. The antioxidant activity of all crude extracts was also investigated by DPPH radical scavenging assay. Crude extract from MFLUCC16-0605 had high antioxidant activity (IC50 value of 15.36 mu g/mL) comparable to the trolox and gallic acid standards showing IC50 values of 2.56 and 12.89 mu g/mL, respectively. The chemical composition of the crude extract from MFLUCC16-0605 was determined using GC-MS. Sixty-two compounds were identified representing 92.09% of crude extract with six major components including 5E,9E-farnesyl acetone, columellarin, totarene, laurenan-2-one, and 8S,13-cedranediol. PCR amplification and sequencing of the barcoding region identified MFLUCC16-0605 as belonging to Nigrospora genus. The notable activities of MFLUCC16-0605 indicate that the endophyte is a potent natural resource and its use as an antibacterial/antioxidant agent should be further explored.