Cholera caused by Vibrio cholerae is a major public health problem among developing countries. The present study is designed to understand the occurrence of V. cholerae using conventional as well as molecular tools and antibiotic susceptibility testing of the isolated strains of V. cholerae from Muttil panchayat in district Wayanad of Kerala, India. This study was undertaken for a period of 12 months i.e., from April 2014 to March 2015. Out of 60 water samples collected from stream, 38 (63.3 %) were found positive for V. cholerae by conventional methods and among these, 21 (35 %) samples were found positive by PCR. However, none of the water samples (well water, household drinking water and pipeline water) yielded V. cholerae. The presence of V. cholerae was observed in the soil and stream from residential area and were also isolated from fish samples as well as from human stool samples. The antibiotic susceptibility testing of the isolates of V. cholerae obtained from different types of samples exhibited 100 % resistance to the antibiotics under study except for doxycycline. Furthermore, on correlating the physical parameters of the water samples collected with the isolation of V. cholerae, it was found that optimum temperature, increased pH and salinity in the present study favoured the survival of V. cholerae, along with high biochemical oxygen demand values of the water samples. The present study provides a baseline data to devise rapid and reliable molecular detection methods for V. cholerae and thereby attaining concrete molecular epidemiological surveillance.