Compared with the venoms of many front-fanged species of snakes (e.g. Viperidae, Elapidae and Atractaspidae) in which the pharmacology, biochemistry, toxinology and mechanism of action of many of the toxins have been fairly extensively studied, the toxic nature and mode of action of the venom toxins of colubrid snakes are less clearly understood. Symptoms associated with the venoms of most medically important colubrid snakes include both diffuse intravascular coagulation and hemorrhage. Although the genus Phalotris occurs from the central region of Brazil down to Paraná State, nothing is known about the composition of its Duvernoy’s gland secretion. Phalotris as well as other members of the sub-family Xenodontinae shows morphological features that suggest a phylogenetical relationship with the Atractaspididae and/or Elapidae families. A reported accident with Phalotris mertensi in Brazil was characterized by cephalea, local and oral mucosa hemorrhage, edema and severe renal insufficiency suggesting the presence of various classes of toxins in the poisonous secretion of this snake. In this work, the complexity of the venom from P. mertensi was examined by various protocols for measuring its in vitro enzymatic activities, and by electrophoresis. Our results show that this venom has proteolytic activity on casein, gelatin and fibrinogen. Determination of its coagulant activity on plasma showed a specific activity 20 times higher than that of Bothrops jararaca venom. Moreover, the venom showed hyaluronidase, phospholipase A2 and myotoxic activities. The general profiles of this venom by 1D-PAGE and 2D-PAGE were similar to the profiles reported in our previous works on Bothrops venoms. 2D-PAGE showed a profile of proteins dispersed across the pI and molecular mass range of the gel. Finally by using specific staining methods applied to 2D-PAGE we examined subpopulations of the venom proteins (metalloproteinase proteome; serine proteinase proteome; phospholipase A2 proteome and the glycoproteome). It is hoped that these results may accelerate research into this colubrid venom and may provide new insights into novel and more efficacious treatments for colubrid envenoming.