The aim of this study was to evaluate the extent of Vibrio cholerae contamination and their antibiotic resistance patterns in sardines, water and phytoplankton in Lake Tanganyika, Tanzania. A total of 120 samples from sardines, water and phytoplankton were collected and analysed for V. cholerae. All isolates confirmed as V. cholerae using Polymerase Chain Reaction were also determined for virulence genes and antibiotic susceptibility. Sardine samples (9%) harboured V. cholerae (non-O1) and water samples (3%) harboured V. cholerae O1. Vibrio cholerae was not detected and isolated in phytoplankton samples. One isolate from water samples harboured both toxin regulatory protein (toxR) and haemolysin gene (hlyA), but not cholera enterotoxin gene (ctx) and toxin co-regulated pilus gene (tcpA). Vibrio cholerae isolates were resistant to Ampicillin (83.33%), Amoxicillin (100%), Chloramphenicol (50%) and Tetracycline (100%). All of the isolates were susceptible to Gentamicin and Ciprofloxacin. The study demonstrated that, fish and water are important reservoirs of V. cholerae. Regardless the absence of ctx and tcpA, constant monitoring for V. cholerae should be done as they pose threat to human health.