This dataset contains the mouse skeletal muscle cross sections stained with immunofluorescence or hematoxylin and eosin (H&E). and the corresponding ROI results from output by MyoAnalyst. Quantifying myofiber size is essential for assessing the health and function of skeletal muscle. While several ImageJ plugins exist for myofiber segmentation and size quantification, they have notable limitaions, including inaccuracy and incompatibility with hematoxylin and eosin (H&E)-stained skeletal muscle cross sections. In this study, we introduce MyoAnalyst, a new ImageJ plugin designed for fully automatic analysis of both immunofluorescence (IF)- and H&E-stained skeletal muscle cross sections. When compared to existing ImageJ plugins, MyoAnalyst demonstrated superior accuracy in myofiber segmentation and boundary recognition for both healthy and injured skeletal muscle cross sections stained with IF. Moreover, as the first ImageJ plugin capable of fully automatical analysis of H&E-stained skeletal muscle cross sections, MyoAnalyst achieved segmentation accuracy comparable to that of IF-stained cross sections. MyoAnalyst features an intuitive graphic interface, enableing automated batch analysis of skeletal muscle cross sections. Its adaptability extends to other applications, such as myofiber type characterization and centralized nuclei analysis. We present MyoAnalyst as an efficient, versatile tool for myofiber quantification, tailored to meet the needs of both researchers and clinicians.