Type III secretion system (T3SS) injectisomes are essential virulence machines that enable Gram negative pathogens to inject effector proteins directly into host cells. While the overall molecular architecture of the injectisome is increasingly well defined, how its cytosolic components dynamically reorganize to regulate secretion remains poorly understood. In particular, whether cytosolic injectisome proteins bind independently or in a coordinated manner, and how these interactions change in response to secretion activating signals and substrate availability, has remained unresolved. Here, we use single-molecule localization microscopy to quantify the injectisome bound times of the cytosolic injectisome proteins SctQ, SctL, and the ATPase SctN in living Yersinia enterocolitica under defined secretion ON and OFF conditions, and in the presence or absence of the secreted effector protein YopE. In secretion OFF states lacking effectors, each cytosolic injectisome protein displays distinct injectisome bound times, consistent with their independent binding kinetics. In contrast, the presence of YopE synchronizes the bound times of all three proteins without itself activating secretion, revealing effector-induced coordination of injectisome binding and unbinding kinetics. Upon secretion activation, a distinct kinetic regime emerges: all three proteins exhibit a subpopulation of long-lived binding events, indicative of cooperative binding behavior. Importantly, both short- and long-lived binding events occur within individual injectisomes – a finding uniquely enabled by super-resolved single-molecule localization measurements. By establishing that secretion state and substrate availability independently modulate and coordinate cooperative protein interactions at injectisomes, our results provide new insights into the dynamic quaternary structure that underpins the function and functional regulation of bacterial type III secretion systems.