Dothistroma needle blight is a forest disease of increasing international importance due to its ability to kill as well as to retard the growth of pines. It is caused by fungi in the genus Dothistroma that produce dothistromin, a non-host selective toxin and virulence factor that is involved in necrosis of pine tissue. Recent studies of the genome of one of the main pathogenic species, Dothistroma septosporum, showed that it contains many similarities to that of the biotroph Cladosporium fulvum, including the presence of candidate biotrophic effector genes, which supported the hypothesis that D. septosporum has a hemi-biotrophic lifestyle. Using Pinus radiata as a host, we used a combination of microscopy, histological and molecular tools to further test this hypothesis and to determine the stage of the disease cycle in which dothistromin toxin is produced. The results showed a biotrophic-type phase in which the fungus grew over the needle surface, penetrated through stomatal pores and colonized epistomatal chambers. The subsequent necrotrophic phase was characterized by colonization of the mesophyll and production of dothistromin, with a >100-fold increase in dothistromin levels from early necrotic lesion to sporulating lesion stages. This is consistent with the role of dothistromin as a virulence factor that is involved in lesion expansion.