Background and Objectives: This study aims to evaluate the association between the use of oral isotretinoin and menstrual irregularities in acne patients with previously regular menstrual cycles. Materials and Methods: A prospective observational study was conducted on 58,599 female patients aged 14 to 36 at King Abdullah University Hospital in Irbid, Jordan. The patients were followed for a period of 4.5 to 8 months during treatment and for 2 months post-treatment. Menstrual cycle changes were documented, and statistical analysis was performed to identify any significant associations. Results: A total of 111 (37.1%) patients, who were previously known to have regular menstrual cycles, complained of menstrual changes while using oral isotretinoin. Ninety-nine of those patients who complained of menstrual changes had their cycles back to normal post-treatment. There is a significant difference in the total accumulative dose between those with changes in menses and those without; p-value [0.008]. The most common change that occurred was amenorrhea (p < 0.001), followed by oligomenorrhea and menorrhagia (p < 0.001 and p = 0.050, respectively). The duration of treatment was a significant predictor of menstrual irregularities, with an odds ratio (OR) of 5.106 (95% CI: 1.371–19.020, p = 0.015), indicating a higher likelihood of menstrual changes with increased treatment duration. The total accumulative dose was also significantly associated with menstrual irregularities (OR = 0.964; 95% CI: 0.939–0.990; p = 0.006). Additionally, a family history of PCOS significantly increased the odds of menstrual irregularities (OR = 3.783; 95% CI: 1.314–10.892; p = 0.014). Conclusions: The study identified that 37.1% of the participants experienced changes in their menstrual cycles while undergoing isotretinoin therapy, with the vast majority (89.2%) returning to normal within two months post-treatment. Our logistic regression analysis pinpointed the duration of isotretinoin treatment, the total accumulative dose, and a family history of PCOS as significant predictors of menstrual irregularities.