Maternal diet may modulate human milk microbiota, but the effects of nutritional supplements are unknown. We examined the associations of prenatal diet and supplement use with milk microbiota composition. Mothers reported prenatal diet intake and supplement use using self-administered food frequency and standardised questionnaires, respectively. The milk microbiota was profiled using 16S rRNA gene sequencing. Associations of prenatal diet quality, dietary patterns, and supplement use with milk microbiota diversity and taxonomic structure were examined using Wilcoxon signed-rank tests and multivariable models adjusting for relevant confounders. A subset of 645 mothers participating in the CHILD Cohort Study (originally known as the Canadian Healthy Infant Longitudinal Development Study) provided one milk sample between 2 and 6 months postpartum and used prenatal multivitamin supplements ≥4 times a week. After adjusting for confounders, vitamin C supplement use was positively associated with milk bacterial Shannon diversity (β = 0.18, 95% CI = 0.05, 0.31) and Veillonella and Granulicatella relative abundance (β = 0.54; 95% CI = 0.05, 1.03 and β = 0.44; 95% CI = 0.04, 0.84, respectively), and negatively associated with Finegoldia relative abundance (β = –0.31; 95% CI = –0.63, –0.01). Fish oil supplement use was positively associated with Streptococcus relative abundance (β = 0.26; 95% CI = 0.03, 0.50). Prenatal diet quality and dietary patterns were not associated with milk microbiota composition. Prenatal vitamin C and fish oil supplement use were associated with differences in the milk microbiota composition. Future studies are needed to confirm our findings and elucidate mechanisms linking maternal supplement use to milk microbiota and child health.