The best time of production of the cyclohexadepsipeptide beauvericin by the endophytic fungus Fusarium oxysporum SS46 in Czapek medium was evaluated. The highest level of beauvericin production was found on day 21 of fermentative culture, as assessed by quantitative analysis by high performance liquid chromatography coupled with a photodiode array detector. Precursor-directed biosynthesis experiments were carried out to produce new analogues of beauvericin by feeding F. oxysporum with ten analogues of L-phenylalanine. In order to evaluate which precursor analogues were incorporated by the microorganism, the obtained extracts were analyzed using matrix-assisted laser desorption ionization - time-of-flight mass spectrometry (MALDI-TOF/TOF). The precursor-directed biosynthesis studies led to the biosynthesis of novel beauvericin derivatives by replacement of one, two, or all three L-phenylalanine residues in beauvericin with DL-3-fluorophenylalanine, L-3-fluorophenylalanine, L-4-fluorophenylalanine, or L-tyrosine. Beyond these precursor analogues, one unit of L-4-aminophenylalanine, L-4-chlorophenylalanine, DL-4-bromophenylalanine, or L-4-bromophenylalanine was also incorporated by the endophyte F. oxysporum SS46. Units of L-4-nitrophenylalanine and L-histidine were not incorporated by the microorganism to produce unnatural beauvericins.