DNA double-strand breaks (DSBs) are among the most lethal forms of DNA damage, repaired primarily by non-homologous end joining (NHEJ) or homologous recombination (HR). While the molecular regulation of these pathways is well characterised, the role of chromatin’s physical state in DSB repair remains poorly understood due to the diffraction limit of light microscopy. Here, using correlative single-particle tracking and phasor histone fluorescence lifetime-Förster resonance energy transfer (histone FLIM-FRET) microscopy, we uncover a novel biophysical mechanism by which BRCA1 prevents chromatin overcompaction and slows DSB mobility, thereby promoting controlled repair. In contrast, 53BP1 promotes chromatin compaction and increases DSB mobility, acting antagonistically to BRCA1. We further show that dual depletion of BRCA1 and 53BP1 restores chromatin mobility to wild-type levels. These findings explain why BRCA1-deficient cancers are sensitive to poly(ADP-ribose) polymerase (PARP) inhibitors, due to increased DSB mobility, and why loss of 53BP1 in these cancers leads to PARP inhibitor resistance by re-establishing normal chromatin dynamics. Our study reveals a previously unrecognised chromatin-based biophysical mechanism underlying PARP inhibitor response in BRCA1-deficient patients.