Aim and objective: The present study determines the incidence of anti-Chlamydia antibodies (ACA), antisperm antibody (ASA), and thyroid levels in serum of infertility patients and statistically determines the significance of all three study parameters. Materials and methods:A cross-sectional study was conducted on volunteers of 190 patients both male and female reporting to the Infertility Clinic of hospital and 178 patients including pregnant women patients were used as control. Serum from patients was collected and analyzed for the presence of anti-Chlamydia antibodies, ASA, serum T3, T4, and thyroid-stimulating hormone (TSH) levels by enzyme-linked immunosorbent assay (ELISA). Results: Infertility patients representing 12.6% of the study population tested positive for anti-Chlamydia antibodies. 21.6% of 190 patients had high ASA levels and these levels were found to be statistically significant p < 0.001 when compared with control. Elevated thyroid levels of the study population observed as increased T3 in 26 patients and T4 in 18 patients with 16 patients having increased TSH and the increased levels of thyroid in infertility patients compared with control is statistically significant p value <0.01. Correlation between all three study parameters is statistically significant [0.01 level (two-tailed)]. Conclusion: Significantly higher levels of anti-Chlamydia antibodies and ASA are found associated in infertile patients as is elevated T3, T4, and TSH. All three parameters together can cause infertility showing the multidimensional factors for the development of infertility. Clinical significance: Patients with infertility should be screened for all three study parameters viz. ACA, ASA, and thyroid levels and subsequent treatment protocols to resolve all possible adverse results to within normal levels for the success of ART. Research highlights• The present study found higher levels of anti-Chlamydia antibody in patients with primary infertility compared with the control group indicating the strong association between chlamydial infections and infertility. • The present study found higher levels of antisperm antibodies in the serum of patients with primary infertility compared with the control group implying a significant role for ASA and development of infertility. • Thyroid disorders such as hypothyroidism due to elevated TSH and hyperthyroidism associated with increased total serum T3 or T4 are several times higher in infertility patients when compared with the general population providing evidence for thyroid imbalance and infertility. • All three study parameters-anti-Chlamydia antibodies, ASA, and thyroid levels occurring together in any one patient may tend to cause infertility.