Laboratory cultivation of Devils Hole pupfish, Cyprinodon diabolis, has proved to be problematic for many years. Although various attempts have been made, long term maintenance and cultivation of this species over multiple generations has thus far been unsuccessful under laboratory conditions. Several strategies are being employed to elucidate the mechanisms behind these difficulties, utilizing hybrid Devils Hole pupfish, C. diabolis x C. nevadensis mionectes as a model. Microbiological and histological analyses are being performed to understand potential pathogens that may affect the physiology of C. diabolis, as well as to design strategies that maximize hatch success. Because many of the bottlenecks previously identified in the captive breeding of Devils Hole pupfish have been during larval rearing, particular effort has been placed on maximizing early survival. Several prophylactic antimicrobial therapies have been compared as they relate to egg hatch rate, larval survival, and long term clearance of targeted pathogens. Treatments of moderate and broad‐spectrum antimicrobials were successful in improving hatch rates and larval survival. Both formalin and iodophor disinfection of pupfish eggs led to better survival of larvae, albeit with slightly reduced hatch rates in the iodine treated eggs. Broad-spectrum antibiotics, particularly trimethoprim sulfamethoxazole and chloramphenicol were especially effective in increasing both hatch rate and juvenile survival. Combinations of formalin plus antibiotic further increased survival, while iodophor disinfection plus antibiotic treatment significantly decreased fifteen-day survival of larvae compared to untreated controls. Results were submitted to the USFWS as a final report, presented in a thesis, and were published in the North American Journal of Aquaculture in December, 2016.