This study evaluated the psychometric properties of the Spiritual Well-Being Scale (SWBS) in a Brazilian sample. We analyzed spiritual well-being, defined as existential well-being (EWB) and religious well-being (RWB), among individuals with varying religious and spiritual experiences, both users and non-users of psychedelics. The online cross-sectional survey was conducted in Brazil, from April to June 2022. The psychometric analyses demonstrated reliability and validity based on the internal structure and the relationship with satisfactory external variables concerning the RWB and EWB factors of the SWBS. Validity evidence was shown for both factors (RWB, EWB) with adequate reliability ratings. However, the RWB factor, which was entirely replicated, demonstrated the best group differentiation and internal consistency. Although both factors showed validity, the RWB factor exhibited superior psychometric indices for validity, group discrimination, and reliability. Regarding psychedelics, the association with RWB and EWB demonstrates a U-shaped pattern, as participants who never use these substances typically exhibit higher RWB and EWB indices, succeeded by frequent users. This finding underscores the need for additional studies to further explore the intricate interplay between psychedelics and spiritual well-being.