The aim of this study was to test the use of mechanical and mechanical-enzymatic methods, saline solution (SS), and PBS solution for the manipulation and isolation of mare ovarian preantral follicles (PAFs). The ovaries were subjected to mechanical isolation (mixer) alone or in association with enzymatic digestion (collagenase). Incubation times of 10 and 20 min were employed. In the first group, 4.1 ± 4.9 PAFs were harvested with the mechanical-enzymatic method vs 71.1 ± 19.2 with the mechanical procedure, showing a significant difference between methods; using SS and PBS, these numbers were 35.7 ± 34.3 and 39.6 ± 39.6, respectively, with no significant difference between solutions. In the second group, there was significant difference between methods, with 7.1 ± 10.6 follicles harvested with the mechanical-enzymatic method vs 63.2 ± 22.9 with the mechanical procedure; using SS and PBS, means were 35.5 ± 36.4 and 34.9 ± 31.1, respectively. The mechanical method proved more effective than the mechanical-enzymatic approach. Both SS and PBS can be used as a media for equine PAFs preparation.