Purpose Orthorexia nervosa (ON) is considered a pathological fixation with healthy eating. Despite recent research focus, there is still a lot of inconsistent information concerning ON, including its definition and validity of its measuring tools. This study aimed to extend the current knowledge on ON, by developing and validating the Portuguese adaptation of the Dusseldorf Orthorexie Scale (DOS) and studying its prevalence and relationship with different psychological indicators, sex, BMI and dietary pattern. Methods Data were collected online, through self-report questionnaires, and two different samples were collected, with 513 (Sample 1) and 541 participants (Sample 2) from the general population. Results DOS’ one-dimensional factorial structure and reliability was confirmed. ON’s prevalence was 10.52%. Results revealed that women present higher scores on DOS than men, but no differences were found regarding the BMI groups. DOS was positively related to all psychological indicators, with moderate and strong relationships with disordered eating and inflexible eating. Sample 2 was used to explore ON’s differences between dietary patterns, with results demonstrating that omnivores present significantly lower levels of orthorexic tendencies, when compared with the vegetarian, vegan, and paleo groups. Conclusions The present study is the first to successfully validate a measure that assesses ON for the Portuguese population and adds to the existing literature in several other important ON aspects. Level of evidence Level V—validation study.