This paper presents the characteristics of bone diagenesis in a secondary commingled Mycenaean burial in Kastrouli (Phocis, Greece) through the histological (light microscopy), physical (FTIR-ATR), and biochemical (collagen) analysis of seventeen human (including two petrous bones) and seven animal bones. Post-mortem modifications in bone microstructure, bioapatite, and collagen were characteristic of burial environments with seasonal groundwater and temperature fluctuations. The two human petrous bones displayed a lack of microscopic focal destruction (MFD) sites and a generally good histological preservation, but although a small sample size, did not show any better bioapatite and collagen preservation compared with human femora. Intra-site variations were defined by three main diagenetic patterns that display differences in histological modifications, crystallinity changes, and collagen degradation. These different patterns were either related to different microenvironment conditions and/or influenced by possible differences in the early taphonomic histories experienced by bones prior to secondary deposition. Further, this study highlights the importance of infrared splitting factor (IRSF), carbonate/phosphate ratio (C/P), and general histological index (GHI) for the qualitative assessment of archaeological bone, and the potential use of amide/phosphate ratio (Am/P) as a collagen predictor.