Background: Chlamydia trachomatis infection is a significant cause of follicular conjunctivitis worldwide, particularly in regions where trachoma is endemic. Aim of the work: This study investigates the prevalence, clinical significance, and molecular diagnosis of Chlamydia trachomatis in patients with follicular conjunctivitis, shedding light on important demographic and clinical associations identifying knowledge gaps and proposing avenues for future research and public health interventions. Methods: Forty patients presenting with follicular conjunctivitis at Ain Shams University hospitals, Egypt. Conjunctival scraping samples were collected, and DNA extraction was performed for amplification targeting the 16S rRNA gene of Chlamydia trachomatis by conventional PCR. Results: The key finding is that 75% of conjunctival samples tested positive for the presence of the C. trachomatis 16S rRNA gene. Significant associations were found between PCR positivity and demographic characteristics such as gender (P < 0.005) with an equal distribution of 20 males and 20 females and age (P = 0.021) ranged from 18 to 64 years, with a median age of 26 years. Clinical symptoms were also strongly associated with positive PCR results (P < 0.005) including redness, foreign body sensation, irritation, itching, tearing, burning sensation, and blurred vision, along with unilateral and/or bilateral site of infection. Conclusion: These results underscore the clinical significance of C. trachomatis in follicular conjunctivitis and stress the necessity for targeted interventions and public health initiatives. Future efforts should focus on the development of a rapid, simple, and cost-effective diagnostic test to curtail the spread of C. trachomatis infection and targeting its proper management.