Eleven strains of the avian pathogen Mycoplasma synoviae were evaluated for the presence of sialidase activity with the use of the fluorogenic substrate 2′-(4-methylumbelliferyl)-α-d-N-acetylneuraminic acid and the sialidase inhibitor 2-deoxy-2,3-didehydro-N-acetylneuraminic acid. The kinetics of in vitro growth in modified Frey medium were also assessed for each strain. Five strains had been isolated from clinically symptomatic chickens, and strains WVU1853^T and K3344 have been demonstrated to be capable of reproducing disease in specific-pathogen-free chickens. All strains exhibited sialidase activity, although the amount varied 65-fold among strains (P < 0.0001) from 1.3 × 10⁻⁷ to 2.0 × 10⁻⁹ activity units per colony-forming unit. Strains originally isolated from clinically symptomatic birds had more (P < 0.05) sialidase activity than strains from asymptomatic birds. Strain WVU1853^T exhibited the most sialidase activity (P < 0.0001) and grew to the highest culture density (P < 0.0001) among strains, but across strains, the rank correlation of growth rate with sialidase activity was not significant. Negligible activity was detected in conditioned culture supernatant fluid. This is the first report of sialidase activity in pathogenic strains of M. synoviae, which suggests a potential enzymatic basis for virulence of the organism.