This study aimed to compare subjective and objective measures of physical activity (PA) in healthcare professionals and assess their adherence to recommended guidelines. A convenience sample of 153 female healthcare professionals (51 nurses, 50 medical caregivers, and 52 physiotherapists) from 24 h healthcare facilities in Poland participated in the study. Subjective assessment was done using an expanded version of the International Physical Activity Questionnaire, while objective assessment involved wearing an Actigraph GT3X accelerometer for seven consecutive days. The results showed no significant correlation between subjective and objective measures of moderate-to-vigorous PA in both occupational and non-occupational activities. Objectively measured PA was significantly lower than self-reported PA, with a large effect size observed for both occupational and total PA. Only 44% of participants met the recommended guidelines for aerobic activity based on accelerometer data, while subjective assessment showed a higher percentage (76%) of healthcare professionals meeting the guidelines. These findings highlight the discrepancy between self-reported and objectively measured PA in healthcare professionals. It emphasizes the need for more accurate assessment methods to understand the actual PA levels of this population. Further research should explore factors influencing PA reporting accuracy and develop interventions to promote regular physical activity among healthcare professionals.