The morphology and dynamics of DNA in a bacterial nucleoid affects the kinetics of such major processes as DNA replication, gene expression. and chromosome segregation. In this work, we have applied fluorescence correlation spectroscopy to assess the structure and internal dynamics of isolated Escherichia coli nucleoids. We show that structural information can be extracted from the amplitude of fluorescence correlation spectroscopy correlation functions of randomly labeled nucleoids. Based on the developed formalism we estimate the characteristic size of nucleoid structural units for native, relaxed, and positively supercoiled nucleoids. The degree of supercoiling was varied using the intercalating agent chloroquine and evaluated from fluorescence microscopy images. The relaxation of superhelicity was accompanied by 15-fold decrease in the length of nucleoid units (from ∼50 kbp to ∼3 kbp).