Bovine milk is a nutrient-dense food and a major component of the human diet. Therefore, understanding the factors that impact its composition is of great importance. Applications of metabolomics provide in-depth analysis of the metabolite composition of milk. The objective of this research was to examine the impact of lactation stage on bovine milk metabolite levels. Metabolomic analysis of bovine milk powder samples across lactation (N = 18) was performed using nuclear magnetic resonance (1H-NMR) spectroscopy and liquid chromatography–tandem mass spectrometry (LC-MS/MS). Forty-one metabolites were identified and quantified in the 1H-NMR spectra. Statistical analysis revealed that 17 metabolites were significantly different across lactation stages (FDR < 0.05), of which the majority had higher levels in early lactation. In total, 491 metabolites were measured using LC-MS/MS, of which 269 had significantly different levels across lactation (FDR < 0.05). Compound classes significantly affected by lactation stage included phosphatidylcholines (59%) and triglycerides (64%), of which 100% of phosphatidylcholines and 61% of triglycerides increased from early lactation onwards. Our study demonstrates significant differences in metabolites across the stages of lactation, with early-lactation milk having a distinct metabolomic profile. More research is warranted to further explore these compositional differences to inform animal feeding practice.