Three new and seven known calopins were isolated from Caloboletus radicans. The structures of the new cyclocalopins, 8-deacetylcyclocalopin B (1), cyclocalopin A-15-ol (2), and 12,15-dimethoxycyclocalopin A (3), were mainly elucidated by NMR and MS data analysis. The stereochemistry of 1–3 was assigned based on NOE correlations and coupling constants and by comparison of their CD spectra with those of similar known calopins. While 1–10 were inactive against two cancer cell lines, they displayed anti-staphylococcal activity against methicillin-resistant Staphylococcus aureus strains (MRSA) with MIC values of 16–256 μg/mL. Moreover, some calopins were active against the fish pathogen Enterococcus faecalis F1B1.