DNA double-strand breaks (DSBs) are the most harmful form of DNA damage. In mammalian cells, two main repair pathways—non-homologous end joining (NHEJ) and homologous recombination (HR)—function in a cell cycle-dependent manner. Key regulators, including BRCA1 and 53BP1, control DNA break-end resection to promote HR and NHEJ, respectively.While the molecular mechanisms of DSB repair are well studied, the role of chromatin’s physical state in repair pathway choice, efficiency, and fidelity remains less understood. To address this, we developed a correlative single-particle tracking (SPT) and histone FLIM-FRET microscopy method to simultaneously assess DNA DSB foci mobility and chromatin structure. We found that BRCA1 and 53BP1 have opposing effects on chromatin dynamics: 53BP1 enhances DSB foci mobility by promoting chromatin compaction, whereas BRCA1 reduces mobility by inducing chromatin decondensation. Notably, knocking down both restores chromatin mobility and compaction to wild-type levels. This provides the first evidence that BRCA1 and 53BP1 play antagonistic roles in controlling chromatin mobility. Since chromatin mobility influences DSB repair efficiency and fidelity, and 53BP1 loss is frequently associated with PARP inhibitor resistance in BRCA1-deficient cancers, our findings offer new insights into the underlying mechanisms of PARPi resistance.