Recently, growing interest is devoted to investigation of bioactive secondary metabolites of endophytic fungi. Thus, as an extension to our previous achievements related to antimicrobial potential of endophytic fungi, Phomopsis species isolated from conifer needles was selected as appropriately promising natural source for drug discovery. Its dichloromethane and ethanol extracts considerably inhibited growth of Escherichia coli and Staphylococcus aureus. Moreover, the individual compounds of dichloromethane extract have been separated, collected and purified using semi preparative liquid chromatographic analysis and comprehensively characterized using mass spectrometry (MS) and nuclear magnetic resonance spectroscopy (NMR). Based on their antimicrobial activity and unique structural characteristics in comparison with well-established drugs from the same therapeutic category, two dominant compounds (Z)-(Z)-2-acetoxyprop-1-en-1-yl-3-(3-((E)-3,4-dihydroxypent-1-en-1-yl)oxiran-2-yl)acrylate (denoted as 325-3) and (Z)-(Z)-2-acetoxyprop-1-en-1-yl 3-(3-((E)-4-hydroxy-3-oxopent-1-en-1-yl)oxiran-2-yl)acrylate (denoted as 325-5) were recognized as valuable leading structures for future discovery of novel antibiotics.