Aim of this study was to determine if Mentofin would have any effect on Mycoplasma gallisepticum (MG) clearance from the tracheal epithelium of chickens in commercial layer flocks, which were naturally infected with MG. Results indicated that, compared to the control group, there was a significant and continuous decline in MG infection in chickens of Mentofin group determined by culture and Real-Time Polymerase Chain Reaction (MGrPCR) (P<0,05). Serology results in the control group indicated an increase in MG positivity from 25% to 40% (P>0,05), while there was no change in the Mentofin group (P>0,05). Culture results for MG positivity decreased from 85% to 5% in the Mentofin group, while this decrease was from 80% to 35% in the control group (P<0,05). There was a prominent decrease from 100% to 20% in MGrPCR positives in the Mentofin group (P<0,05) compared to a non-significant change observed from 95% to 80% in the control group (P>0,05). Results of this study indicate that Mentofin clearly had an effect on MG clearance from the tracheal epithelium, supported by detection of decline in MG infection in layers.