Prostate-specific antigen blood testing has improved early detection of prostate cancer (PCa); however, PCa mortality has not decreased accordingly and a prostate biopsy is still required for a definitive diagnosis. Proteomic biomarker screening in easily available body fluids such as seminal plasma is now increasingly being proposed as a solution to improve PCa detection and prognosis. PCa cells typically produce high levels of reactive oxygen species (ROS). In this study, we therefore investigated ROS levels in semen samples from patients with a negative or a positive prostate biopsy to predict PCa diagnosis. Multiple clinicopathological parameters (digital rectal examination, prostate-specific antigen scoring, prostate biopsy, and ROS levels) of patients examined for PCa were measured. No significant differences in ROS levels were detected in relation to PCa diagnosis. Although seminal plasma is a well-suited medium for prostate-related biomarkers, no significant differences in ROS levels were observed between the patient groups. Comparison with ROS levels encountered in semen of larger patient groups is the next logical step.