Although DBP (di-n-butyl phthalate) is commonly encountered as an artificially-synthesized plasticizer with potential to impair fertility, we confirm that it can also be biosynthesized as microbial secondary metabolites from naturally occurring filamentous fungi strains cultured either in an artificial medium or natural water. Using the excreted crude enzyme from the fungi for catalyzing a variety of substrates, we found that the fungal generation of DBP was largely through shikimic acid pathway, which was assembled by phthalic acid with butyl alcohol through esterification. The DBP production ability of the fungi was primarily influenced by fungal spore density and incubation temperature. This study indicates an important alternative natural waterborne source of DBP in addition to artificial synthesis, which implied fungal contribution must be highlighted for future source control and risk management of DBP.