Purpose: Fluctuations in female sex hormones result in changes in the gingival and periodontal tissues. The purpose of this study was to compare the periodontal status of premenopausal women at different time points during their menstrual cycle and to find the associated subgingival microbiota. Materials and Methods: One hundred premenopausal women participated in the study and were divided into two groups: group I consisted of 50 subjects with clinically healthy gingival, and group II consisted of 50 subjects with chronic gingivitis. Group II was further divided into group IIa and group IIb. Group IIa consisted of 25 subjects who did not receive any periodontal therapy during the study period. All the examinations were performed at three points during the menstrual cycle: ovulation (OV), pre-menstruation (PM) and menstruation (M). Plaque Index (PI), Gingival Index (GI), Papillary Bleeding Index (PBI), probing depth (PD), subgingival temperature (ST) recording, gingival crevicular fluid (GCF) collection, and estimation and microbiological examination using the benzoyl-DL-arginine-naphthylamide (BANA) test was carried out. For group IIb subjects, all the examinations were performed again during the next menstrual cycle, which followed 4 weeks after periodontal therapy. Results: Women with clinically healthy gingiva exhibited negligible changes throughout the menstrual cycle, whereas women with gingivitis showed aggravated inflammation during ovulation and pre-menstruation as compared to menstruation. However, there was no alteration in subgingival microbiota. After treating gingivitis, the next menstrual cycle following 4 weeks after periodontal therapy was monitored, and no periodontal changes were detected. Conclusion: Ovarian hormones have a negligible effect on clinically healthy periodontium. However, these hormones may exaggerate pre-existing inflammation in gingival tissues, but the clinical significance of these changes remains uncertain. Keywords: gingival crevicular fluid, gingivitis, inflammation, menstrual cycle, periodontium, sex steroid hormones