Persistent organic pollutants (POPs) are toxic chemical compounds that can bioaccumulate, adhere to lipid matrices, and affect human health. This study aimed to investigate the association between maternal pre-pregnancy overweight/obesity and dietary intake during pregnancy and POP concentrations in the human milk of women from Rio de Janeiro, Brazil. One hundred and forty-seven women were followed from the third trimester of pregnancy until 119days postpartum, and 77 human milk samples were analyzed between 2 and 119days postpartum. POP concentrations were analyzed using gas chromatography-triple quadrupole mass spectrometry. Pregnancy dietary intake was estimated using a semi-quantitative food frequency questionnaire, and pre-pregnancy body mass index at baseline was classified as normal or overweight/obesity. Multiple logistic and linear regression models were performed to investigate the association between pre-pregnancy overweight/obesity, dietary intake during pregnancy, and POP concentrations in human milk. The models were adjusted for maternal age, maternal schooling, total cholesterol serum concentrations, and time postpartum. The analyses were corrected for multiple comparisons using the Benjamini-Hochberg test. Significant associations were observed between pre-pregnancy overweight/obesity and dichlorodiphenyldichloroethane (ppDDE), polychlorinated biphenyl (PCB)74, PCB138, PCB153, PCB170, PCB180, total PCBs, total 4PCBs, total 2 organochlorine pesticides (OCPs), and total POP concentrations. Higher daily lipid intake during pregnancy increased human milk hexachlorobenzene (HCB). This study showed that pre-pregnancy overweight/obesity and total lipid intake during pregnancy were associated with POP concentrations in the milk of women from Rio de Janeiro, Brazil. To promote adequate nutritional status since preconception and surveillance and control of POP in the environment could be essential to ensure binomial mother-infant health and biomonitoring studies and programs for these POPs should be stimulated.