Gossypol interferes with reproduction, causing damage to sperm, disrupting the estrous cycle and resulting in embryonic lethality. In females, gossypol administration promotes degeneration of ovarian follicles, but it is unknown whether this effect is direct or indirect. Thus, the aim of this study is to determine whether gossypol interferes with folliculogenesis in vitro in rats, mice and goats. Ovaries from rats and mice and fragments of goat ovaries were grown in cell culture for 24 h or 7 days. Four groups were tested: 0 (control), 5, 10 and 20 μg gossypol/ml. After incubation, the ovaries were fixed and processed for histological analysis. Follicles were classified according to their stage of development as either viable or atretic. It was found that the ovaries of rats, mice and goats cultured with gossypol showed an increase in the proportion of atretic follicles and a consequent reduction in the proportion of viable follicles at all stages of follicular development. Compared to the control group, the viability of all ovarian follicles in the rat, mouse and goat groups was reduced after cultivation for 24 h by 56.9%, 56.5% and 68.0%, respectively, with the highest concentration of gossypol (20 μg/mL), and after seven days, the respective reductions were 65.4%, 65.3% and 88.2%. Thus, it is possible that gossypol may directly affect follicular maturation, and consequently female fertility.