Selenium is involved in many metabolic pathways that are critical for life. Information concerning the metabolic effects of selenium in autism spectrum disorder (ASD) and obesity is still conflicting and incomplete. The pre- and post-pubertal selenium profiles of patients with ASD and obesity have not yet been investigated. The goal of the study was to examine selenium content before and after puberty in euthyroid children diagnosed with ASD, compared to age-matched neurotypical controls, with respect to overweight or obesity as a co-existing pathology. Serum, toenail, and 24h urine selenium levels were determined by inductively coupled plasma mass spectrometry in 287 prepubertal children (mean age 8.09 years), divided into groups: ASD with overweight/obesity (ASD+/Ob+); ASD without overweight/obesity (ASD+/Ob−); non-ASD with overweight/obesity (ASD−/Ob+); and non-ASD without overweight/obesity (ASD−/Ob−). The assessment was repeated in 258 of the children after puberty (mean age 14.26 years).The lowest serum (p < 0.001), urine (p < 0.001) and toenail (p < 0.001) selenium levels before and after puberty were observed in ASD+/Ob+ patients, and the highest in ASD−/Ob−. There were no differences in serum/toenail selenium levels between ASD+/Ob− and ASD−/Ob+ groups. The presence of ASD was associatedwith lower serum (p < 0.001) and toenail (p < 0.001) selenium in BMI-matched groups. In neurotypical patients, post-pubertal serum selenium levels were lower (p < 0.001) than pre-pubertal levels. In the multiple linear regression analyses, selenium levels showed inverse relationships with BMI (p < 0.001) and male gender (p < 0.001), irrespective of the sample type. The serum (p = 0.002) and toenail (p < 0.001) selenium levels were inversely associated with the presence of ASD. ASD, obesity/overweight, and male gender have independent impacts on selenium levels in children. Puberty may affect selenium content in neurotypical children of both genders, but not in ASD patients.