The hierarchical chromatin organization begins with formation of nucleosomes, which fold into chromatin domains punctuated by boundaries and ultimately chromosomes. In a hierarchal organization, lower levels shape higher levels. However, the dependence of higher-order 3D chromatin organization on the nucleosome-level organization has not been studied in cells. We investigated the relationship between nucleosome-level organization and higher-order chromatin organization by perturbing nucleosomes across the genome by deleting Imitation SWItch (ISWI) and Chromodomain Helicase DNA-binding (CHD1) chromatin remodeling factors in budding yeast. We find that changes in nucleosome-level properties are accompanied by changes in 3D chromatin organization. Short-range chromatin contacts up to a few kilo–base pairs decrease, chromatin domains weaken, and boundary strength decreases. Boundary strength scales with accessibility and moderately with width of nucleosome-depleted region. Change in nucleosome positioning seems to alter the stiffness of chromatin, which can affect formation of chromatin contacts. Our results suggest a biomechanical “bottom-up” mechanism by which nucleosome distribution across genome shapes 3D chromatin organization.