Aims: To isolate, characterize and determine the antibacterial activities of compounds produced by the endophytic fungus Diaporthe sp. F2934, cultivated on malt extract agar. Methods and Results: The fungus was cultivated aseptically in Petri dishes containing malt extract agar at 25 degrees C for 15 days. Crude extract was obtained from mycelium using ethyl acetate and sonication, and was fractioned using classic chromatography and HPLC. The structures of phomosines and chromanones were established by NMR experiments including HMQC, HMBC and COSY. Their molecular formulas were determined by ESI-TOFMS. We obtained six compounds: (1) 4H-1-benzopyra-4-one-2,3-dihydro-5-hydroxy-2,8-dimetyl, (2) 4H-1-benzopyran-4-one-2,3-dihydro-5-hydroxy-8-(hydroxy-lmethyl)-2-methyl, (3) 4H-1-benzopyra-4-one-2,3-dihydro-5-methoxyl-2,8-dimetyl, (4) phomosine A, (5) phomosine D and (6) phomosine C. Isolated compounds 1, 2 and 5 were inactive against 15 micro-organisms, but phomosines A and C were active against diverse Gram-negative and Gram-positive bacteria. Conclusions: A group of new chromanones and known phomosines have been isolated from the genus Diaporthe (Diaporthe sp. F2934). The results obtained confirm the wide chemical diversity produced by endophytic fungi, specifically the genus Diaporthe. In addition, phomosines A and C may be considered as antimicrobial agents that can be used to guide the development of new antibiotics. Significance and Impact of the Study: Our phylogenetic analysis places Diaporthe sp. F2934 as sister to the Diaporthe cynaroidis clade. Three chromanones were isolated and identified, for the first time, using crude extract obtained from Diaporthe F2934. From this extract phomosines A, C and D were also purified. Regarding Staphylococcus aureus, the inhibition zone diameter (IZD) for phomosine A was 20% higher than the standard drug, vancomycin. When cultivated as described here, Diaporthe sp. F2934 produced new and antimicrobial compounds.