In regions like Qatar, where access to fresh water is limited, unlicensed artesian wells are often a source of water for agricultural and livestock purposes These wells are mostly unlicensed and therefore do not come under the supervision of the responsible authorities in the state for water quality control and determining their suitability for irrigation or drinking. However, due to the absence of regulation and monitoring, these wells are prone to bacterial contamination, posing significant health risks. This study focuses on the assessment and development of biotechnological methods for detecting bacterial contaminants in unlicensed artesian wells around Doha. Utilizing a combination of microbiological and molecular techniques, such as pour plating, chromogenic assays, and multiplex PCR, we identified bacterial species including E. coli, Bacillus subtilis, and Serratia marcescens. Our findings indicate a substantial presence of coliform bacteria in many of the tested water samples, confirming the risk posed by these unregulated wells. This investigation underscores the importance of routine testing and proposes the implementation of accessible testing methods to ensure water safety and protect public health. Recommendations for future research and policy development are provided to enhance water management practices in similar arid regions.