Salmonella Gallinarum,is a host specificpathogenic bacterium of fowl typhoid, one of the most important diseases of poultry that increases the death rate and reduction in eggs production. Bacteria forms the complex structural colonies enclosed in a sticky matrix known as biofilm. Various antimicrobial approaches used to treat gastrointestinal infections are usually ineffective due to biofilm formation. The purpose of this study was to (1) compare biofilm formation of three S. Gallinarum strains isolated from commercial broiler chicken by three different methods i.e., congo red, test tube and air liquid interface coverslip, (2) biofilm quantification at different time intervals and (3) monitor antibiofilm effect of three proteolytic enzymes including trypsin, chymotrypsin and proteinase k against S. Gallinarum. We observed that S. Gallinarum has strong biofilm forming ability as observed by dark black colonies on congo red medium. Quantification assays such as test tubes revealed significantly (p<0.001) strong biofilm after 5 days with significantly increased planktonic cells (after 3 days) and increased loosely bound cells (after 5 days). Similarly, air liquid interface coverslip indicated significant increase in biofilm after 1 day. Comparison of antibiofilm effect using proteolytic enzymes indicated that although all enzymes resulted in significant decrease (p<0.05) in biofilm formation after 1 hour, however, inhibitory effect of proteinase k was more pronounced (p<0.001; 80%) compared to the other two enzymes (45% and 34 % respectively). Hence, we concluded from this study, that S. Gallinarum is a strong biofilm former. Use of proteases can strongly inhibit biofilm formation in vitro and can be used as effective therapeutic approach to control fowl typhoid epidemic along with an antibiotic therapy. The future prospects of the current study may include the testing of these proteases in poultry feed to see their effect on S. Gallinarum pathogenesis in vivo.