Abstract Enzymatically active macrodomains of (+)ss-RNA viruses mediate immune evasion by countering ADP-ribosylation and are therefore promising druggable targets. Here we report testing of ADP / ADP-ribose analogues for their ability to inhibit Mac1 of SARS-CoV-2, measurement of the affinity of active compounds and characterization of their binding mode by cocrystallization, uncovering critical molecular determinants of protein-ligand interaction. Key findings of the resulting structure-activity relationship (SAR) include that inhibitory potency is improved by either replacing the distal ribose of ADP-ribose by a small alkyl group or the adenine N7 by carbon. Based on insights from the SAR, we show β-methyl-GS-441524-diphosphate as nanomolar inhibitor that exhibits >1000-fold selectivity over human MacroD1 and MacroD2. Addition of C 11 -acyloxybenzyl (AB)-masking groups yields a membrane permeable, lipophilic prodrug that inhibits SARS-CoV-2 in cell culture (EC 50 0.06 µM) while exhibiting low cytotoxicity (CC 50  > 50 µM). Replacement of the terminal methyl phosphate with an ethyl phosphonate increases stability of the prodrug with little effect on toxicity and antiviral potency (EC 50  = 0.03 µM), making it a membrane-permeable nucleotide-based prodrug against viral macrodomains.