Background: Chlamydia trachomatis (CT) is a sexually transmitted infection that requires early detection to prevent complications. This study aimed to evaluate the prevalence of CT among asymptomatic women in Spain and investigate the relationship between CT and risk factors associated with sexual practices, as well as factors such as stress and depression. Results: We found that 3.8% of asymptomatic women tested positive for CT. Our findings suggested that having more than five sexual partners increases the risk of sexually transmitted infections (STIs) by 3.87 times when compared with having fewer partners (p = 0.005, OR: 3.87, 95% CI 1.24–11.65). Additionally, 4.5% of participants admitted to using drugs. We found that there was a slightly higher proportion of anxiety and depression among women who tested positive for CT. Conclusions: We aimed to establish a basis for the implementation of screening in asymptomatic women. Early identification and preventive measures are crucial in minimizing the long-term complications and transmission of the disease. Sexual behavior must be recognized as a risk factor, and women’s psychological well-being should be given top priority as a vital aspect of their sexual health.