The gold standard for diagnosing infectious diseases is culture-based identification of bacterial pathogens, which is time-consuming and labour-intensive. Current advances in molecular diagnostics and microfluidic technologies have made the rapid detection of bacteria or viruses in clinical specimens possible. However, the need for rapid, sensitive and multiplex detection of pathogens in a “sample-in and answer-out” manner has not been fully satisfied. In this study, a self-contained and fully integrated fluidic cassette and its supporting analyser were constructed for multiplex detection of bacteria to accelerate the diagnosis of urinary tract infections (UTIs). The fully integrated cassette contains all the necessary components and reagents for bacterial analysis. All of the bacterial analysis processes, including bacterial lysis, magnetic silica bead-based DNA extraction, DNA elution and multiplex loop-mediated amplification (LAMP), are automatically conducted in the cassette. This cassette was successfully applied for the detection of four major pathogenic bacteria in UTIs, i.e., Escherichia coli, Proteus mirabilis, Salmonella typhimurium and Staphylococcus aureus. The first three were successfully detected with a limit of detection (LoD) of 1 colony-forming unit (CFU) μL⁻¹ and the last was with a LoD of 10 CFU μL⁻¹ in urine samples, demonstrating that the cassette has similar sensitivity compared to that of the manual protocol, which is lower than that required by UTIs. The turnaround time for this cassette-based sample-to-answer system was approximately 100 minutes, and the detection is sensitive, fully automated, and accurate, demonstrating the potential to be a useful diagnostic tool for UTIs.