•Microfluidisation induced molecular unfolding and rearrangement of almond proteins.•α helix reduced by 11 % & β sheets by 24 % at 80 MPa pressure compared to control.•80 MPa pressure significantly improved almond protein’s digestibility & PDCAAS.•Almond meal proteins functionality improved with increasing pressure up to 80 MPa.•High pressure >80 MPa reversed beneficial functional properties of almond proteins. Almond protein isolate (API) obtained from almond meal was processed using dynamic high-pressure microfluidisation (0, 40, 80, 120, and 160 MPa pressure; single pass). Microfluidisation caused significant reductions in the particle size and increased absolute zeta potential. SDS-PAGE analysis indicated reduction in band intensity and the complete disappearance of bands beyond 80 MPa. Structural analysis (by circular dichroism, UV–Vis, and intrinsic-fluorescence spectra) of the API revealed disaggregation (up to 80 MPa) and then re-aggregation beyond 80 MPa. Significant increments in protein digestibility (1.16-fold) and the protein digestibility corrected amino acid score (PDCAAS; 1.15-fold) were observed for the API (80 MPa) than control. Furthermore, significant improvements (P < 0.05) in the functional properties were observed, viz., the antioxidant activity, protein solubility, and emulsifying properties. Overall, the results revealed that moderate microfluidisation treatment (80 MPa) is an effective and sustainable technique for enhancing physico-chemical and functional attributes of API, thus potentially enabling its functional food/nutraceuticals application.