Two sialylated human milk oligosaccharides (SHMOs) 3'-sialyllactose (3'-SL) and 6'-sialyllactose (6'-SL) were accessed for their possible antiviral activity against six different subtypes of thirteen avian influenza (AI) viruses in vitro. 3'-SL exhibited promising antiviral activity against almost all subtypes of tested AI viruses in hemagglutination inhibition assay, whereas 6'-SL showed activity against few selected H1N1, H1N2, and H3N2 subtype strains. 3'-SL has minimum inhibitory concentration values of 15.62 mM or less in more than half of the viruses examined. 3'-SL also showed effective inactivation of H9N2 Korea isolate (A/Chicken/Korea/MS96/1996) at 12.5 mM concentration in Madin Darby Canine Kidney (MDCK) cell line. Thus, 3'-SL was further studied for in vivo study against H9N2 virus in pathogen free chicken experiment models. In vivo study exhibited improved clinical symptoms on H9N2 infected chickens when treated with 3'-SL. Moreover, treating chickens with 3'-SL resulted in complete elimination of H9N2 viruses within 24 h of virus infection (0.8 HAU of H9N2). Indirect ELISA assay confirmed complete wash-out of H9N2 viruses from the colon after neutralization by 3'-SL without entering the blood stream. These in vivo results open up possible applications of 3'-SL for the prevention of AI virus infections in birds by a simple cleansing mechanism.