Some recent researches have shown the important role of hair cortisol as a retrospective biomarker of chronic stress. The aim of this research was to investigate the relationship between hair cortisol levels and sociodemographic and psychological variables, such as perceived stress levels and psychopathological symptoms on a Spanish population. The sample consisted of 347 healthy people, 230 women and 117 men, with an average age of 33.39 years (SD = 12.63). Hair cortisol levels were measured by obtaining a hair sample. In addition, a psychological assessment composed by: Analogic-Visual Stress Scale, Perceived Stress Scale (PSS), Symptom Checklist 90 Revised (SCL–90–R) and the assessment of vital stressful events suffered, was carried out. The mean cortisol level was 108.93 pg/mg (SD = 66.43) in men, and 120.38 pg/mg (SD = 87.26) in women. The linear hierarchical regression showed that Analogic-Visual Stress Scale and perceived stress levels were related with higher hair cortisol levels (R2 = .032; t = 2.21; p = .029). Due to the relationship between daily stress levels, Analogic-Visual Stress Scale, anxiety sub-scale of SCL 90–R and perceived stress levels with hair cortisol levels, we conclude that there is a relation between perceived yourself stressed and the physiological levels.