We evaluated the effectiveness of four antibiotics in enhancing the hatch rate, larval survival, and adult survival of hybrid Devils Hole Pupfish Cyprinodon diabolis (hybridized with Ash Meadows Amargosa Pupfish C. nevadensis mionectes). Cephalexin (CEX; concentration = 6.6 mg/L of water), chloramphenicol (CAM; 50 mg/L), erythromycin (ERY; 12.5 mg/L), and trimethoprim sulfamethoxazole (TMP-SMX; 25 mg/L) were applied as a constant bath either to incubating eggs or to larvae that hatched from untreated eggs. Hatch rate was roughly doubled by incubation in the presence of CAM (68% hatch) and TMP-SMX (66%) relative to the control (28%). Cephalexin and ERY conferred no benefit upon the hatch rate. Among fry that hatched from treated eggs, there was no increase in 15-d larval survival. However, fish that hatched from eggs treated with CAM, ERY, and TMP-SMX demonstrated enhanced survival at 360 d (51.2, 38.4, and 43.6%, respectively) and at 540 d (22.6, 6.8, and 20.2%, respectively); the untreated control had no survivors to those time points. All groups of eggs treated with antibiotics showed reductions in bacterial colony-forming units (CFUs) at 24 h posttreatment. At 120 h posttreatment, CEX-treated eggs had CFU counts similar to those of the control, whereas the TMP-SMX-treated eggs had the lowest CFU counts. Eggs treated with CAM and ERY had similar CFU counts, which were significantly reduced from the control counts. Larvae that were treated with CAM and TMP-SMX within 12 h posthatch showed enhanced 15-d survival (74% and 72%, respectively) in comparison with the control (56%). For pupfish rearing efforts in which antibiotic use is appropriate, CAM and TMP-SMX appear to provide the greatest benefit, particularly when applied to incubating eggs rather than to hatched larvae.