MSc. (Molecular Biology and Biotechnology)Toxigenic Vibrio cholerae O1 and O139 are responsible for the production of cholera toxin and subsequently cause cholera. Although cholera has been very rampant around African great lakes, little is known about the status of V. cholerae in aquatic reservoirs like fish. This study aimed to determine the occurrence of toxigenic and drug resistant V. cholerae in Tanganyika Tilapia (Oreochromis tanganicae) and water from Lake Tanganyika in Tanzania. Repeated cross-sectional study design that involved 140 Tilapia fish samples and 60 water samples was carried out in Lake Tanganyika during dry and rain seasons. Bacterial analyses culture and biochemical tests were used for identification of V. cholera. Positive isolates were confirmed by PCR of the ompW and ctxA genes then serotyped with polyvalent O1 antiserum. Antimicrobial susceptibility patterns of V. cholerae O1 (n = 65) were performed using disk diffusion method. Results of the study revealed a prevalence of V. cholerae at 27.9% (n = 39/140) in Tanganyika tilapia and 33.3% (n = 20/60) in water samples. The proportions of toxigenic V.cholerae in Tanganyika Tilapia and water were 11.4% (n = 16/140) and 20% (n = 20/60) respectively. Prevalence of toxigenic V. cholerae in fish gills and intestines were 5.7% (n = 16/280) and 0.4% (n = 1/280) respectively. Vibrio cholerae were susceptible to Gentamicin (100%), Ciprofloxacin (100%), Cefotaxime (100%), Ceftazidime (73.8%), Chloramphenicol (63.1%), Tetracycline (53.8%) and Naliadixic acid (47.7%). Resistance in V. cholerae was observed for Streptomycin (100%), Amoxicillin (95.4%), Ampicillin (81.5%), Sulphamethaxole (80%) and Trimethoprim (72.3%). This study revealed that Tanganyika Tilapia and water in Lake Tanganyika harbor toxigenic, antibiotic resistant V. cholerae of the epidemic potentials, with high incidences in wet season. Raising awareness on public health practices (hygiene/sanitation) and rational use of antimicrobial agents to safeguard public health from cholera outbreaks are recommended.