Six fungi strains (M. anisopliae 3935, 4516, 4819, PL57, PL43 and M. flavoviride CG291) were studied regarding their ability to produce an anticancer indolizidine alkaloid. The culture process was carried out in Shaken flask at 26ºC and 200 rpm using three different culture medium containing oat meal extract supplemented with glucose and DL-lysine or Czapek culture medium. The mycelial extracts produced by Metarhizium spp. cultures were directly submitted to electrospray ionization mass spectrometry (ESI-MS) analysis and the highest alkaloid concentration (approximately, 6 mg.L-1) was reached when M. anisopliae 3935 was tested.