Purpose: Hormonal fluctuations, primarily in progesterone and estrogen during the menstrual cycle, may influence periodontal tissues, with inflammatory cytokines playing a crucial role. Therefore, our primary objective was to assess clinical periodontal parameters and measure levels of interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α in gingival crevicular fluid (GCF) throughout the menstrual cycle. Methods: This longitudinal prospective study was conducted from February to April 2022 and included 50 participants. We assessed clinical periodontal parameters—plaque index (PI), gingival index (GI), pocket depth (PD), clinical attachment loss (CAL), and tooth mobility—at three stages of the menstrual cycle: menstruation day, ovulation day, and premenstrual day. Additionally, GCF samples were collected using paper points. These samples were then stored and analyzed for levels of IL-1β, IL-6, and TNF-α using enzymelinked immunosorbent assays. Results: There were 25 participants in our study. The GI, PD, and CAL increased significantly during the menstrual cycle and were significantly higher during the premenstrual phase than in the ovulation phase (P<0.05). The levels of GCF IL-1β (P=0.012), IL-6 (P=0.002), and TNF-α (P=0.015) showed statistically significant throughout the menstrual cycle compared to baseline which was the menstrual (follicular) phase. Furthermore, the GCF levels of IL-1β and IL-6 reached their peak during the luteal or premenstrual phase, whereas TNF-α peaked during the ovulation phase. The increase in biological markers was more pronounced between the menstruation phases than the clinical periodontal markers. Conclusions All clinical periodontal parameters, except for the PI, showed a slight increase from the follicular phase to the luteal phase, with significant differences observed between each phase. The levels of GCF IL-1β (P=0.012), IL-6 (P=0.002), and TNF-α (P=0.015) were statistically significant, with increases in IL-1β and IL-6 throughout the menstrual cycle, peaking in the luteal phase. This demonstrates the influence of the menstrual cycle on clinical periodontal and GCF inflammatory markers.