Simultaneous and quantitative assessment of multiple tissue parameters may facilitate more effective diagnosis and therapy in many clinical applications, such as wound healing. However, existing wound assessment methods are typically subjective and qualitative, with the need for sequential data acquisition and coregistration between modalities, and lack of reliable standards for performance evaluation or calibration. To overcome these limitations, we developed a multimodal imaging system for quasi-simultaneous assessment of cutaneous tissue oxygenation and perfusion in a quantitative and noninvasive fashion. The system integrated multispectral and laser speckle imaging technologies into one experimental setup. Tissue oxygenation and perfusion were reconstructed by advanced algorithms. The accuracy and reliability of the imaging system were quantitatively validated in calibration experiments and a tissue-simulating phantom test. The experimental results were compared with a commercial oxygenation and perfusion monitor. Dynamic detection of cutaneous tissue oxygenation and perfusion was also demonstrated in vivo by a postocclusion reactive hyperemia procedure in a human subject and a wound healing process in a wounded mouse model. Our in vivo experiments not only validated the performance of the multimodal imaging system for cutaneous tissue oxygenation and perfusion imaging but also demonstrated its technical potential for wound healing assessment in clinical practice.