This present work describes the application of liquid chromatography-solid phase extraction-nuclear magnetic resonance spectroscopy to analyse Alternaria alternata crude extracts. Altenusin (1), alternariol (2), 3'-hydroxyalternariol monomethyl ether (3), and alternariol monomethyl ether (4), were separated and identified. High-resolution mass spectrometry confirmed the proposed structures. The cytotoxic effects of these compounds towards plants were determined using soybean (Glycine max) cell cultures as a model. EC(50) values which range from 0.11 (± 0.02) to 4.69 (± 0.47) μM showed the high cytotoxicity of these compounds.