Early-life exposure to natural and/or synthetic chemicals can impact acute and chronic health conditions. Here, a suspect screening workflow anchored on high-resolution mass spectrometry was applied to elucidate xenobiotics in breast milk and matching stool samples collected from Nigerian mother-infant pairs (n = 11) at three-time points. Potential correlations between xenobiotics and the gut microbiome, as determined by 16S rRNA gene amplicon sequencing, were subsequently explored. Overall, 12,192 and 16,461 features were acquired in the breast milk and stool samples, respectively. Following quality control and suspect screening, 562 and 864 features remained, respectively, with 149 of these features present in both matrices. Taking advantage of 242 authentic reference standards measured for confirmatory purposes of both, potentially beneficial and adverse xenobiotics, 34 features in breast milk and 68 features in stool were unambiguously identified and subsequently semi-quantified. Moreover, 51 and 78 features were annotated with spectral library matching, as well as 416 and 652 within silicofragmentation in breast milk and stool, respectively. Despite that the workflow was originally optimized for polyphenols, a diverse range of other chemical classes were simultaneously identified including mycotoxins, endocrine-disrupting chemicals (EDCs), antibiotics, plasticizers, perfluorinated alkylated substances, and pesticides. Spearman rank correlation of the identified features revealed significant correlations between chemicals of the same classification such as polyphenols. One-way ANOVA and differential abundance analysis of the data obtained from stool samples revealed that features of plant-based origin were elevated when complementary foods were introduced to the infants’ diets. Features in the stool deemed significant by ANOVA, such as tricetin, positively correlated with the genusBlautia. Moreover, vulgaxanthin negatively correlated withEscherichia-Shigella. Despite the limited sample size, this exploratory study provides high-quality exposure data of matched biospecimens obtained from mother-infant pairs in sub-Saharan Africa, and showed potential correlations between the chemical exposome and the gut microbiome.Graphical abstract