This study explores the role of pre-colonized microbial cultures in enhancing the long-term effectiveness of microbial electrochemical sensors for water quality monitoring. Microbial electrochemical sensors rely on specific functional microorganisms to detect and signal changes in environmental water quality. Pre-colonization of these cultures on the sensor’s electrode can promote sustained detection sensitivity. This study investigates how specific microbial groups— Geobacter species, sulfate-reducing bacteria (SRB), and acetogen microbes—affect sensor performance when initially enriched and transferred to the biosensor. In the pre-enrichment phase, microbes were incubated in three defined media: Geobacter medium, SRB medium, and acetogen medium. Each culture was cycled through three 48 h incubation periods to establish dominant microbial populations and then introduced to independent biosensors. A control sensor was seeded with natural inoculum from Tasek Kejuruteraan UKM. The results showed that Geobacter -enriched biosensors quickly generated strong electrical signals by oxidizing substrates at the anode, marking them as the most effective at facilitating electron transfer. SRB-enriched sensors produced negative signals, as SRB consumed electrons and thrived at the cathode. Acetogen -enriched biosensors exhibited slower, indirect electron transfer, with lower electrochemical activity. In contrast, the control sensor displayed only minimal increases in signal strength over time. The Geobacter -enriched biosensor, which achieved a significant current drop from 0.478 mA to 0.093 mA (an ~80.5% decrease) upon pollutant exposure, demonstrated the fastest response to rising pollutant levels, followed by SRB, acetogen, and the control. These findings emphasize the importance of starting with targeted microbial populations to optimize biosensor functionality for environmental monitoring applications. IMPORTANCE Microbial electrochemical sensors are widely recognized as effective tools for environmental monitoring and water quality assessment. Numerous studies have explored the enrichment and adaptation of microbial communities in various environmental conditions, focusing on their interactions, survival, and metabolic performance. However, a critical gap remains largely overlooked—specifically, the importance of the biosensor start-up procedure and the selection of initial microbial populations. The presence of specific electrogenic bacteria at the sensing terminal during start-up plays a vital role in initiating and sustaining biosensor functionality. In this study, we aim to address this gap by not only examining the performance of the biosensor system itself but also emphasizing the role of pre-enriched microbial communities. Our approach focuses on building a healthy, functional, and responsive biosensing platform by optimizing microbial colonization from the onset.