Biscoumarin derivatives 1-27 were tested for their inhibition of snake venom and human nucleotide pyrophosphatase phosphodiesterase-1 enzymes. Lineweaver-Burk and Dixon plots and their secondary replots showed that these compounds are pure non-competitive inhibitors of both the enzymes. Ki and IC50 values of biscoumarins were found to be in the range of 50 to 1000 and 164 to > 1000 microM, respectively, against human recombinant phosphodiesterase 1 enzyme and 8.0 to 1150 and 9.44 to > 1000 microM, respectively, against snake venom phosphodiesterase. Compounds 1, 3, 4, 6, 7, 17, 26, and 30 were found to be non-competitive and non-cytotoxic upto a concentration of 200 microg/mL as evident by less than 10% cell death after 3 h of incubation.