The study evaluated the effect of iron supplementation for maternal anemia on fetal fraction (FF) by gestational age (GA) in 308 pregnant women undergoing NIPT. There was a moderate positive correlation between GA and FF (ρ = 0.399, p < 0.001), while hemoglobin (Hb) levels showed a weak positive correlation with FF (ρ = 0.112, p = 0.050). Median FF was significantly higher after 16 weeks of gestation (11.7% vs. 8.3%, p < 0.001). After week 16, FF was significantly lower in women with iron supplementation compared to those without (p = 0.010), before week 16 the difference was not significant. In multivariable regression analysis adjusted for GA, Hb, and maternal anemia, GA remained the only independent factor associated with FF. Crude analyzes suggested a lower FF after iron supplementation after week 16, but multivariable analysis showed that these were differences related to GA and hematological parameters, not a direct effect of therapy.