Physical activity is beneficial to health, leading to improvements in outcomes such as blood pressure, cholesterol levels, and adiposity measurements. However, no research has examined the extent to which health professionals are familiar with the physical activity guidelines. This study aimed to: examine the knowledge of Physical Education or other health professionals (Biomedicine, Biological Sciences, Dentistry, Nursing, Physiotherapy, Medicine, Nutrition, Psychology), regarding the guidelines for physical activity and sedentary behavior. A convenience sample of health professionals with higher education training, including physicians, physical education professionals, nutritionists, physiotherapists, nurses, and others, was invited to participate in this study. Individuals holding dual higher education degrees in Physical Education and another health-related field were excluded. The questionnaire consisted of 30 specific questions, based on World Health Organization Guidelines for physical activity and sedentary behavior. No significant differences (p > 0.05) were observed in the overall knowledge scores of the physical activity guidelines between the Physical Education professionals’ group (19.0 ± 5.9 points) and the health professionals’ group (18.0 ± 5.8 points). Significant differences were found between groups in scores for questions about children, adults, the elderly, pregnant women, and postpartum women (p = 0.0140), with Physical Education professionals scoring 4.4 ± 2.2 points and other health professionals scoring 3.6 ± 1.5 points. This research highlights Brazilian health professionals' awareness of the WHO physical activity recommendations, while also pointing to the importance of strengthening education and training to ensure that health professionals are adequately prepared to advocate for physical activity.