Hair analysis of endocannabinoids and N-acylethanolamines has emerged as a promising methodological advancement for the retrospective assessment of cumulative long-term endocannabinoids and N-acylethanolamines secretion in biopsychological research of stress-related symptoms and disorders. In this study, we investigated the influence of the potential associated factors on hair endocannabinoid and N-acylethanolamine levels in 760 adult participants aged between 21 and 86 years old. Gender, age, medication intake, drug use, body mass index, waist circumference, smoking status, alcohol consumption and perceived stress were assessed through questionnaires. Hair endocannabinoid and N-acylethanolamine concentrations were measured through liquid chromatography coupled to tandem mass-spectrometry. Results identified that gender, age, BMI, waist circumference, medication and drug use, hair washing frequency as potential confounding variables of hair endocannabinoid and N-acylethanolamine levels, while no associations were observed with respect to hair dyeing, smoking status, alcohol consumption and perceived stress. The present data indicate that hair endocannabinoids and N-acylethanolamines measurement may be a useful alternative to the current circulating endocannabinoids measurements in biopsychological research with the consideration of possible confounders.