We studied the role of three glycosidases (α- mannosidase - α-ΜΑΝ, β-N-acetyloglucosaminidase - NAGASE and β-galactosidase - β-GAL) in follicular fluid (FF) and in fertilization medium (FM) of bovine oocytes. Oocytes were allocated into 3 groups according to the follicular size (controls - CF: 2-8 mm, small follicle group - SF: 2-5 mm, large follicle group - LF: >5-8 mm). Bovine embryos were produced in vitro either in groups (experiment 1, n = 2099 oocytes) or individually (experiment 2, n = 79 oocytes). In both experiments, the activity of all glycosidases in the FF of large follicles was significantly lower than in the FF of small follicles group. In the FM of LF-group oocytes, α- MAN and NAGASE were significantly higher compared to SF- and CF-group oocytes (experiment 1) and β-GAL was significantly higher in SF- compared to CF-group oocytes (experiment 2). Cleavage rate was similar among all groups in both experiments; however significantly higher blastocyst formation was noted in CF-group compared to LF- (days 7, 8, 9) and SF- (days 8, 9) groups (experiment 1). In follicular fluid of small follicle group, β-GAL was associated positively with degenerating oocytes’ number and negatively with blastocyst rate at days 7, 8 (P = 0.065) and 9 (experiment 1). In fertilization medium of control group, α-MAN related negatively to cleavage rate (P < 0.05) and β-GAL to blastocyst rate at day 8 (P = 0.089) or day 9 (P = 0.072) (experiment 1). During fertilization, in experiment 1 all oocytes consumed β-GAL and only control or small follicle oocytes consumed α-MAN; in experiment 1, only large follicle oocytes released NAGASE, whereas all oocytes released all three glycosidases in experiment 2. In conclusion, glycosidases affect the developmental competence of oocytes collected from different sized follicles during in vitro fertilization, performed either in groups or individually; their role in follicular fluid is different from that in fertilization medium.