This study was conducted to investigate the effect of full-fat oil seeds on milking performance, milk composition, and milk quality in lactating Holstein cows. Animals for the experiment were randomly assigned to one of three treatment groups. The control group was fed a CON diet (total mixed ration + hay + concentrate), T1 was fed the CON diet + 600 g flaxseed, and T2 was fed the CON diet + 600 g flaxseed + 200 g chia seeds. Supplementation with flaxseed or flaxseed + chia seeds had little effect on milk yield, feed intake, and feed efficiency of lactating Holstein cows. The content of protein and nonfat-solids was higher in T1 than in control (P < 0.05) during the first period, but overall, the addition of flaxseed or flaxseed + chia seeds had a low influence on milk composition. Conjugated linoleic acid (CLA) and polyunsaturated fatty acid contents increased with the supplementation of flaxseed or flaxseed + chia seeds (P < 0.05). The percentage of n-3 fatty acids and the antioxidant activity of raw milk also increased as a result of flaxseed or flaxseed + chia seed administration (P < 0.05). Thus, in this study, mixed flaxseed and chia seed supplementation exerted a positive effect on lactation persistency, functional fatty acid concentration (CLA, polyunsaturated fatty acid, and n-3 fatty acid), and antioxidant activity in lactating Holstein cows.