Abstract: The inhibition effects of an antagonistic Bacillus strain B96-II on asparagus stem blight (Phomopsis asparagi Sacc.) were investigated under laboratory in vitro and field conditions. The results demonstrate that B96-II effectively inhibits the pathogen. Inhabitation rates on P. asparagi growth rate are between 97.37% and 92.98%, when the cultures treated with 50 to 1 600 times dilution of B96-II fermentation brew. Under in vitro conditions, colonial growth on asparagus stem reduces by 94.40% compared with the control samples, and under field trials by 93.40%. Microscopic observation reveals that the mycelia and spore walls of P. asparagi are destroyed and cell contents released after B96-II treatment. Conductivity of the inoculum liquid of P. asparagi increases significantly 24 h after treatments. As B96-II inoculum liquid concentration increases from 1% to 10%, its conductivity increment increases from 47.50% to 518.33% and total dissolved solids (TDS) increment increases from 176.10% to 797.60%. B96-II also restricts P. asparagi mycelial respiration at an inhibition rate of 25.00% by addition of 1% B96-II and 196.40% by addition of 10% B96-II. Based on the analysis, the main active substances in B96-II are antagonistic proteins.