Interaction between HPV and C. trachomatis (CT) in cervical carcinogenesis is not well understood. We studied the joint effects of HPV and CT on the risk of developing high-grade CIN or adenocarcinoma in situ (AIS). In a cohort of 94,349 women with prediagnostic paired serum samples, 490 women developed cervical CIN3/AIS. Serum IgG antibodies to HPV6/16/18/31/33/45 and CT were measured by ELISA. Control group consisted of a subcohort of 2976 women without CIN3/AIS. The cutoff antibody levels for different HPV types were predefined. CT antibody level was defined as negative or positive. Rate ratios (RR) and variances were estimated by Cox model. Since we were interested in the risk associated with the temporal order of infections, the reference group was dual seropositivity at the first serum sampling. When comparing the temporal exposure, we noted high RR of CIN3/AIS for incident CT and hrHPV clade A7 (HPV18/45) infection (RR 25, 95% CI 3.8, 170) compared to the reference group. Concomitant incident infection with HPV18 and CT or HPV45 and CT was associated with very high risk for CIN3/AIS. Adjustment for HPV16 had no effect on the point estimates. The high risk associated with incident (within on average 2.4 years) CT and HPV18/45 infections is in line with the HPV16- independent role of CT in the development of CIN3/ICC shown before. In conclusion, the temporal order of infections has an effect on the development of CIN3/AIS. The risk associated with concomitant CT and HPV18/45 infection suggests that HPV vaccination and screening for hrHPV and CT play significant roles in cervical cancer prevention.