The microfibril angle (MFA) of crystalline cellulose in the wood cell wall along the stem axis has major effects on stiffness and longitudinal shrinkage of wood and is of key importance to timber quality. The aims of this study were: (1) to develop partial least square (PLS) regression models for microfibril angle (measured on tangential sections by X-ray diffraction) based on NIR spectra measured on tangential and on radial surfaces; (2) to develop PLS regression models for MFA based on radial NIR spectra collected from wood surfaces of different quality; and (3) to verify the reliability of these PLS-R models by external validations. T values were recorded by X-ray diffraction on tangential sections while NIR spectra were taken on tangential and radial wood surfaces. PLS-R calibrations for MFA based on tangential NIR spectra were better (r2p = 0.72) than those using radial NIR spectra (r2p = 0.64). The key role of the chemical components and the effect of surface quality of wood on NIR spectroscopy calibrations are discussed. Considering the differences between experimental conditions, these findings showed the potential of the NIR-based models for predicting MFA in Eucalyptus wood, even using spectra taken from different wood faces and surface qualities.