Purpose: To examine the association between muscle fiber typology and match running performance in professional Australian football (AF) athletes. Methods: An observational time–motion analysis was performed on 23 professional AF athletes during 224 games throughout the 2020 competitive season. Athletes were categorized by position as hybrid, small, or tall. Athlete running performance was measured using Global Navigation Satellite System devices. Mean total match running performance and maximal mean intensity values were calculated for moving mean durations between 1 and 10 minutes for speed (in meters per minute), high-speed-running distance (HSR, >4.17 m·s−1), and acceleration (in meters per second squared), while intercept and slopes were calculated using power law. Carnosine content was quantified by proton magnetic resonance spectroscopy in the gastrocnemius and soleus and expressed as a carnosine aggregate z score (CAZ score) to estimate muscle fiber typology. Mixed linear models were used to determine the association between CAZ score and running performance. Results: The mean (range) CAZ score was −0.60 (−1.89 to 1.25), indicating that most athletes possessed a greater estimated proportion of type I muscle fibers. A greater estimated proportion of type I fibers (ie, lower CAZ score) was associated with a larger accumulation of HSR (>4.17 m·s−1) and an increased ability to maintain HSR as the peak period duration increased. Conclusion: AF athletes with a greater estimated proportion of type I muscle fibers were associated with a greater capacity to accumulate distance running at high speeds, as well as a greater capacity to maintain higher output of HSR running during peak periods as duration increases.