Zidovudine (ZDV) has antibacterial activity against many members of the family Enterobacteriaceae, including Salmonella species, and may be responsible for a decrease in the frequency of salmonellosis in persons infected with human immunodeficiency virus (HIV). Other nucleoside analogs, such as didanosine (2',3'-dideoxyinosine [ddI]) and zalcitabine (2',3'-dideoxycytidine [ddC]), which have undefined anti-salmonella activity, increasingly are being used in the treatment of HIV infection. To evaluate the anti-Salmonella activity of the antiviral agents ZDV, ddI, ddC, and acyclovir (ACV), we determined MICs for 39 nontyphoidal Salmonella blood isolates. ZDV (MIC for 50% of strains tested [MIC50], 0.5 microgram/ml; MIC range, 0.125 to 4 micrograms/ml) and ddI (MIC50, 8 micrograms/ml; MIC range, 2 to 125 micrograms/ml) had concentration-dependent activity. Anti-Salmonella activity was not observed for ddC or ACV. Nine Escherichia coli blood isolates were inhibited by ZDV (MIC50, 0.125 microgram/ml; MIC range, 0.031 to 1 microgram/ml) to a greater degree than they were by ddI (MIC50, 62.5 micrograms/ml; MIC range, 31 to > 62.5 micrograms/ml). Inoculum size affected susceptibility to ZDV and ddI for Salmonella and E. coli isolates. Resistance to ZDV or to ddI could be induced in vitro in Salmonella isolates, but cross-resistance was not observed. These results indicate that at concentrations achieved during the treatment of HIV infection, ZDV has activity against nontyphoidal salmonellae, although resistance can develop. ddI, ddC, and ACV at currently used dosages would not be expected to be effective in the prevention or treatment of Salmonella infections.