Background and Objectives: Orienteering is a sport characterized by high physical exertion and intense mental demands, which increase susceptibility to errors. Understanding the impact of such errors on psychophysiological responses, particularly on heart rate variability (HRV), is essential. This study aimed to investigate the relationship between psychophysiological indicators and checkpoint errors made by elite orienteers during official competition. Materials and Methods: Fifty-three orienteers participated in this study, and their performance was continuously monitored and recorded by using a global positioning system (GPS) and HRV data. Errors made during the orienteering events were identified and analyzed. HRV data were examined in three temporal segments: before, during, and after the identified and standardized errors. Results: The analyses indicated that errors significantly impacted HRV indices across multiple domains: the time domain, frequency domain, and nonlinear domain. Additionally, a significant effect of sex on the normalized the root mean square of successive differences (r-MSSD) before and after the error was observed. Conclusions: The findings of this study underscore the significant impact of errors made by orienteers on cardiovascular responses, as evidenced by measurable alterations in HRV metrics. Cardiovascular activity, represented by the HRV, can provide useful information for coaches and sport psychologists to adopt effective training programs for athletes.