<p>The classic psychedelic psilocybin is receiving renewed interest in naturalistic and clinical research settings. Despite trends indicating increased rates of use outside of research settings, longitudinal data on the association between psilocybin use, mental health, and other substance use remains limited. A prospective longitudinal online survey study comprising six sequential assessments from adults planning to take psilocybin in naturalistic settings was conducted. A total of 2,850 respondents completed the survey 2&#xa0;weeks before they planned to use psilocybin, 1,551 completed the 2–4-week follow-up, and 657 completed the 2–3-month follow-up after psilocybin use. A repeated measures latent profile analysis of internalizing problems, substance use, and cognitive flexibility was conducted. The repeated measures latent profile analysis identified a four-profile model as best fitting the data provided across the three measurement periods: “Improved Mental Health with Low Substance Use” (30.25%), “Stable Mental Health with Low Substance Use” (53.12%), “Persistent Mental Health Symptoms with Persistent Substance Use” (8.84%), and “Improved Mental Health with Persistent Substance Use” (7.79%). Sociodemographic and personality characteristics prior to psychedelic use were associated with profile membership, with the profiles also differing significantly on measures of the acute subjective effects of psilocybin. Although psilocybin use was associated with improvements in internalizing problems for the majority of participants, persisting mental health difficulties and substance use problems were also noted. These findings highlight the heterogeneous associations between psilocybin use, mental health, and patterns of other substance use in non-clinical settings.</p>