This repeated cross-sectional study assessed the validity and reproducibility of the myfood24® dietary assessment tool against dietary intake biomarkers in healthy Danish adults. The study included 71 healthy adults (14/57 m/f), aged 53.2 ± 9.1 years with an average BMI of 26.1 ± 0.3 kg/m2. Participants were instructed to complete seven-day weighed food records using myfood24® at baseline and 4 ± 1 weeks thereafter. Estimated mean dietary intake was compared with objective measures of energy metabolism and selected dietary intake biomarkers in fasting blood (folate) and in 24-hour urine (urea, potassium). Resting energy expenditure was measured by indirect calorimetry. Application of the Goldberg cut-off classified 87% (n = 62) of participants as acceptable reporters. A strong Spearman’s rank correlation was observed between total folate intake and serum folate (ρ = 0.62). Acceptable correlations were noted for serum folate (ρ = 0.49) and urinary potassium excretion (ρ = 0.44) with estimated and measured protein intake (ρ = 0.45); energy intake and total energy expenditure (ρ = 0.38); potassium intake and potassium excretion (ρ = 0.42); and estimated fruit and vegetable intake. Reproducibility analysis revealed strong correlations (ρ ≥ 0.50) across most nutrients and food groups, except for fish and vitamin D (ρ = 0.30 and ρ = 0.26, respectively). Notably, reproducibility for folate and total vegetable intake exhibited the highest correlations (ρ = 0.84 and ρ = 0.78, respectively). In conclusion, while some limitations exist, myfood24® remains a useful tool for ranking individuals by intake, particularly in studies focusing on relative comparisons.