Commercial aviation pilots are an occupational group that work in particular conditions, with frequent schedule changes, shift work, unfavorable environmental conditions, etc. These circumstances can lead to fatigue, work overload (WO), and daytime sleepiness, factors that can affect their health and safety. This study aimed to assess the prevalence and the association between these parameters in a sample of Spanish commercial airline pilots. The Raw TLX, Fatigue Severity Scale, and the Epworth Sleepiness Scale questionnaires were administered in a sample of 283 participants. The relationships of the total scores between all the questionnaires were studied by the chi-square test and the risk scores (odds ratio) were calculated. Different models using multiple linear regression were carried out to evaluate the effects of WO, fatigue, and daytime sleepiness, among the total scores, age, and flight hours. Additionally, the internal consistency of each questionnaire was estimated. A total of 28.2% presented WO above the 75th percentile, with mental and temporal demand the dimensions with the greatest weight. A total of 18% of pilots presented fatigue, 15.8% moderate sleepiness, and 3.9% severe sleepiness. We observed an association among WO, fatigue, and daytime sleepiness, important factors related to pilot health and aviation safety.