Presence of pyrethroid insecticides in human breast milk and in thatch wall material of dwellings from Southern Africa subtropical area (Manhiça, Mozambique) was investigated to assess potential pyrethroid route of human exposure. Human breast milk samples were collected during 2002 when pyrethroids were widely used as insecticides for mosquito bed nets in Mozambique for malaria control. The median concentration value of total pyrethroids ranged between 87 and 1200 ng/g lw, with λ-cyhalothrin being the most predominant pyrethroid in human breast milk contributing for 35% of the total amount. Moreover, and for the first time, an isomer-specific enrichment was found in human breast milk, showing a selective isomeric accumulation or metabolism in the human body. Based on the calculated pyrethroid concentrations in human breast milk, the daily ingestion rate of pyrethroid was estimated. The nursing infant dietary intake ranged from 0.67 to 9.0 μg (kg of body weight)− 1 day− 1. In addition, thatch materials collected after the reintegration of dichlorodiphenyltrichloroethene (DDT) as insecticide residual spraying (IRS) in Mozambique, showed the presence of pyrethroids with concentration values ranging between 6.9 and 700 ng/g dw. In thatch material as well as in human breast milk, pyrethroid contamination was mainly attributed to the agriculture usage of this insecticide knowing that agriculture represent the 80% of the economy in Mozambique. However, a possible usage of this insecticide as IRS in Mozambique cannot be excluded despite their low efficiency for malaria control. The continued use of these compounds (both for agriculture and malaria prevention) and the ingestion rates calculated from the breast milk concentrations indicate that these insecticides cannot be overlooked for the assessment of the lactation risks of breastfeeding infants from the Manhiça region.