Mid-infrared spectroscopy (MIRS) is traditionally used for analysing milk fat, protein and lactose concentrations in dairy production, but there is growing interest in using it to predict difficult, or expensive-to-measure, phenotypes on a large scale. The resulting prediction equations can be applied to MIRS data from commercial herd-testing, to facilitate management and feeding decisions, or for genomic selection purposes. We investigated the ability of MIRS of milk samples to predict milk fatty acids (FAs) and energy balance (EB) of dairy cows in Australia. Data from 240 Holstein lactating cows that were part of two 32-day experiments, were used. Milk FAs were measured twice during the experimental period. Prediction models were developed using partial least-square regression with a 10-fold cross-validation. Measures of prediction accuracy included the coefficient of determination (R2cv) and root mean-square error. Milk FAs with a chain length of ≤16 were accurately predicted (0.89 ≤ R2cv ≤ 0.95), while prediction accuracy for FAs with a chain length of ≥17 was slightly lower (0.72 ≤ R2cv ≤ 0.82). The accuracy of the model prediction was moderate for EB, with the value of R2cv of 0.48. In conclusion, the ability of MIRS to predict milk FAs was high, while EB was moderately predicted. A larger dataset is needed to improve the accuracy and the robustness of the prediction models.