Edible and medicinal mushrooms are widely studied for their bioactive compounds, yet their role as sources of essential vitamins remains inadequately defined and often overestimated. This review provides a critical assessment of vitamin composition in edible and medicinal mushrooms, with an emphasis on B-group vitamins and vitamin D
2, focusing on variability, bioavailability, and limitations for nutritional applications. Current evidence indicates that mushrooms can contribute to the intake of selected B vitamins, particularly riboflavin (B
2) and thiamine (B
1), at levels comparable to common plant foods. However, their relevance as a source of vitamin B
12 is highly uncertain due to pronounced compositional variability, the frequent occurrence of inactive corrinoid analogues, and limited evidence of physiological bioavailability. In contrast, vitamin D
2 represents a distinctive and technologically controllable feature of mushrooms, formed via the ultraviolet-induced conversion of ergosterol. Post-harvest UV exposure can substantially enhance vitamin D
2 content, enabling targeted biofortification strategies. Nevertheless, the nutritional significance of mushroom-derived vitamins is constrained by inconsistencies in reported concentrations, lack of standardized analytical methodologies, and insufficient clinical evidence. Overall, edible and medicinal mushrooms should not be regarded as universal natural sources of vitamins; rather, their nutritional relevance depends on species, cultivation conditions, post-harvest processing, analytical verification, and, particularly in the case of vitamin D
2, controlled UV-induced biofortification. Future research should prioritize standardized analytical approaches and well-designed human studies to support evidence-based nutritional applications.
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