The aim of this study was to investigate associations between human exposure to persistent organic pollutants and sex hormone levels. The study population (n = 253) was a subsample of GraMo adult cohort, recruited in 2003–2004 in two hospitals from Granada, Spain. Exposure was estimated by analyzing samples of adipose tissue (3 organochlorine pesticides and 3 polychlorinated biphenyls) and serum (4 per- and polyfluoroalkyl substances). Data analyses included robust linear regression and weighted quantile sum regression. In men, PFNA (positively) and PCB-138 (negatively) were associated with testosterone. In premenopausal women, OCPs and PCBs were negatively associated with estradiol and SHBG. PFHxS was inversely related to luteinizing hormone. Additionally, PCB-153 and -180 was positively associated with testosterone. The mixture of PCBs/OCPs was positively associated with testosterone in women and with FSH in men. Our results highlight the potential of chemical mixture exposure to alter sex hormone homeostasis, depending on sex and menopausal status.