This chapter discusses the soft microfluidic scanning electrochemical microscopy (SECM) probes represent a versatile tool for supporting various investigations ranging from the electrochemical characterization of different surfaces to the local perturbation of the microenvironment of adherently grown live cells. Microfluidic SECM probes were electrochemically characterized by cyclic voltammetry (CV) in a redox mediator solution delivered through the microfluidic channel. SECM in the feedback mode has been employed for the imaging of human fingerprints based on the detection of silver nanoparticles or benzoquinone-tagged proteins. The application of the microfluidic SECM probes as a tool for spatiotemporal live cell perturbations has a significant potential toward biological applications, for monitoring in vitro hypoxic tumor models toward the discovery of new therapeutic compounds and the understanding of effects induced by localized pH changes within malignant cells. The microfluidic SECM probes can be easily multiplexed in order to investigate large surfaces or increase the capacity toward drug screening.