Background: Omega-3 fatty acid supplementation has been shown to help maintain muscle function and reduce muscle soreness after exercise-induced muscle damage. However, the relationship between the dietary and biological status of omega-3 fatty acids in rugby players remains unclear. Objectives: This study aimed to investigate the connection between the dietary and biological status of omega-3 fatty acids in university rugby players. Methods: We conducted a cross-sectional study involving university rugby players and age-matched sedentary controls. We assessed diets, including omega-3 and omega-6 polyunsaturated fatty acids, using self-administered dietary questionnaires. Whole blood lipidomics was performed before and after a single training session. Results: The rugby group (n = 29) had significantly higher intakes of omega-3 and omega-6 fatty acids compared to the control group (n = 31). While the blood omega-6 relative concentration did not differ between the groups, the rugby group had lower omega-3 fatty acid levels than the control group (4.4 ± 1.1 vs. 6.2 ± 1.8%). Approximately 48% of rugby players had an omega-3 index (O3i) considered as high risk for cardiovascular disease, and 52% were classified as intermediate risk. None of the players had a low-risk O3i. A single training session had an impact on the omega-3 and omega-6 metabolic pathways. Conclusions: Despite having a higher omega-3 fatty acid intake than controls, rugby football players may still be deficient in omega-3 fatty acids. Further nutritional interventions are needed to address these issues.