•Simultaneous measurement of superoxide and MMP in stallion spermatozoa is described.•Measured values in the superoxide/MMP assay did not correlate with other parameters.•Multivariate analysis revealed differences in contributions to principal components.•The combined superoxide/MMP measurement provides additional information. An improved fertility prediction for stallions is of importance for equine breeding. Here, we investigate the potential of a combined staining of stallion spermatozoa for superoxide and mitochondrial membrane potential (MMP) for this purpose. Semen samples were analysed immediately after arrival at the laboratory, as well as after 24 h. Superoxide was measured by MitoSOXRed, while MMP was measured with JC-1. Menadione was used to stimulate superoxide production. In addition, other parameters of sperm quality, namely motility, membrane integrity, chromatin integrity, sperm kinematics and Hoechst 33258 exclusion were measured and correlated to superoxide production and MMP. Both bivariate correlations between measured parameters as well as multivariate analysis were performed. Measured values in the superoxide/MMP assay did not correlate with other parameters. However, there was a strong negative correlation (r = 0.96 after 0 h, r = 0.95 after 24 h) between membrane integrity and chromatin integrity. Moderate positive correlations were found between motility parameters and membrane integrity, as well as moderate negative correlations between motility parameters and chromatin integrity. The multivariate analysis revealed that membrane integrity, chromatin integrity and motility contributed to the first principal component, while the second was influenced by superoxide/MMP parameters as well as sperm kinematics. Storage of samples for 24 h decreased motility, chromatin integrity and membrane integrity. In conclusion, combined measurement of superoxide and MMP provides additional information not obtained by other assays of sperm quality.