This study aimed to compare the activity of selected glycosidases (β-galactosidase, α-l-fucosidase, β-N-acetyl-hexosaminidase, and sialidase) in homogenates of uterine tissues obtained from female dogs with and without pyometra. In addition, it examined the availability of substrates for these glycosidases in the homogenates. The study was carried out on female dogs undergoing ovariohysterectomy for pyometra (n = 10) and clinically healthy dogs (n = 10) undergoing elective spaying. The activity of β-galactosidase, α-l-fucosidase, and β-N-acetyl-hexosaminidase was analyzed using a spectrofluorometer and that of sialidase using a colorimetric method. Sodium dodecyl sulphate–polyacrylamide gel electrophoresis with Alcian Blue (AB) and Periodic Acid–Schiff (PAS) staining was performed to determine the presence of substrates for these glycosidases in the homogenates of uterine tissues. The results revealed that the activity of all the examined glycosidases was significantly higher (P < 0.05) in the uterine tissues isolated from dogs with pyometra in comparison to healthy dogs. The electrophoretic patterns of the selected samples showed several proteins, which contained different sugar moieties stained by AB and PAS and the profiles differed significantly between the pyometra group and the healthy group. Densitometric analysis of AB staining showed patterns between 233 and 148, 86 and 55, and 43 and 20 kDa, which differed markedly in sugar content between the examined groups of animals. Similarly, PAS staining analysis revealed patterns of different molecular weights, between 233 and 117 and between 55 and 32 kDa, which also differed in sugar content. These findings suggest that canine pyometra is accompanied by the increase in the activity of selected glycosidases in the uterus. This could potentially modify the glycan structures of uterine glycoproteins and in result their biological functions. Further studies are needed to elucidate the potential role of the increased activity of glycosidases in the pathogenesis of this disease.