Chronic respiratory disease (CRD) is one of the major problems within the poultry farms. In this study, a polyvalent inactivated M. gallisepticum and Escherichia coli vaccine was prepared then evaluated using both serological and vaccination challenge tests. Results of this vaccine was promising, where the peak of humoral immune response against M. gallisepticum as measured by ELISA were 2418 and 5600 when taken in either single or booster doses respectively while were 2240 and 3576 for E. coli. As regards to the protection against challenge with virulent bacterial strains, the achieved protection percentages against M. gallisepticum virulent strain were 62 and 72 post single and booster doses while were 66.67 and 77.47 for E. coli. The achieved results could help to be concluded that the locally prepared polyvalent M. gallisepticum and E. coli vaccine may help and minimize the problem of the chronic respiratory diseases in poultry farms.