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.