Contamination of surface waters with pathogens as well as all diseases associated with such events are a significant concern worldwide. In recent decades, there has been a growing interest in developing analytical methods with good performance for the detection of this category of contaminants. The most important analytical methods applied for the determination of bacteria in waters are traditional ones (such as bacterial culturing methods, enzyme-linked immunoassay, polymerase chain reaction, and loop-mediated isothermal amplification) and advanced alternative methods (such as spectrometry, chromatography, capillary electrophoresis, surface-enhanced Raman scattering, and magnetic field-assisted and hyphenated techniques). In addition, optical and electrochemical sensors have gained much attention as essential alternatives for the conventional detection of bacteria. The large number of available methods have been materialized by many publications in this field aimed to ensure the control of water quality in water resources. This study represents a critical synthesis of the literature regarding the latest analytical methods covering comparative aspects of pathogen contamination of water resources. All these aspects are presented as representative examples, focusing on two important bacteria with essential implications on the health of the population, namely Pseudomonas aeruginosa and Escherichia coli.