Ecumicin and rufomycin 4-7 disrupt protein homeostasis in Mycobacterium tuberculosis by inhibiting the proteolytic activity of the ClpC1/ClpP1/ClpP2 complex. Although these compounds target ClpC1, their effects on the ATPase activity of ClpC1 and proteolytic activity of ClpC1/ClpP1/ClpP2 vary. Herein, we explored the ClpC1 molecular dynamics with these compounds through fluorescence correlation spectroscopy. The effect of these compounds on the ATPase activity of ClpC1-cys, the recombinant protein for fluorescence labeling, and proteolytic activity of ClpC1-cys/ClpP1/ClpP2 were identical to those of native ClpC1, whereas the intermolecular dynamics of fluorescence-labelled ClpC1 were different. Treatment with up to 1 nM ecumicin increased the population of slower diffused ClpC1 components compared with ClpC1 without ecumicin. However, this population was considerably reduced when treated with 10 nM ecumicin. Rufomycin 4-7 treatment resulted in a slower diffused component of ClpC1, and the portion of this component increased in a concentration-dependent manner. Ecumicin can generate an abnormal ClpC1 component, which cannot form normal ClpC1/ClpP1/ClpP2, via two different modes. Rufomycin 4-7 only generates slower diffused ClpC1 component that is inadequate to form normal ClpC1/ClpP1/ClpP2. Overall, we demonstrate that ecumicin and rufomycin 4-7 use different action mechanisms to generate abnormal ClpC1 components that cannot couple with ClpP1/ClpP2.