Sports nutrition is an evolving field, but there is a lack of data on Australian athletes’ knowledge of current sports nutrition guidelines. Additionally, several tools used to assess nutrition knowledge (NK) have not undergone adequate validation. The purpose of this study was to assess and compare the sports NK of elite and nonelite Australian football (AF) athletes using a newly validated questionnaire—The Nutrition for Sport Knowledge Questionnaire. Elite AF players (n = 46) were recruited directly from their club dietitian and nonelite AF players (n = 53) were invited to participate via e-mail from their club president or secretary. The mean NK score of elite and nonelite AF players was 46 ± 16% and 51 ± 11%, respectively (p = .041). In both groups, knowledge of macronutrients, weight management, and alcohol was better than knowledge of supplements, micronutrients, and sports nutrition. Nonelite athletes achieved statistically significantly higher scores on the questionnaire subsections testing weight management (elite: 48 ± 18; nonelite: 57 ± 19, p = .019), micronutrients (elite: 39 ± 19; nonelite: 50 ± 16, p = .004), and alcohol (elite: 52 ± 13; nonelite: 71 ± 17, p = .002). While overall NK of Australian athletes was poor, scores varied greatly among individuals (range: 10–70%) and across the six subsections (topics) being assessed. Professionals working with athletes should undertake an assessment of the athletes’ NK so that they can provide targeted education programs.