Denitrification is a critical microbial process for nitrogen removal in wastewater treatment, offering a cost-effective and sustainable alternative to conventional chemical and physical methods. This review synthesizes current knowledge on the microbial ecology of denitrification, focusing on the diversity, physiology, and community dynamics of denitrifiers in biofilms and activated sludge systems. Key bacterial genera, including Pseudomonas, Paracoccus, Hyphomicrobium, Comamonas, and Azoarcus, play dominant roles, with carbon sources such as methanol, ethanol, acetate, and waste-derived substrates strongly shaping community structure and function. Advances in molecular approaches—such as PCR-based techniques, stable isotope probing, fluorescence in situ hybridization, metagenomics, and transcriptomics—have provided new insights into microbial diversity, gene expression, and metabolic pathways, linking ecological patterns with treatment performance. Applications of denitrification span conventional activated sludge processes, biofilm reactors, and emerging autotrophic methods such as anammox, which enhance nitrogen removal efficiency. Despite these advances, operational challenges remain, including incomplete denitrification, seasonal failures, greenhouse gas emissions, and limited predictability of microbial responses to environmental shifts. Integrating molecular data into process models and optimizing carbon source utilization represent key strategies for future improvement. This review highlights the opportunities and challenges in bridging microbial ecology with engineering practices, ultimately advancing wastewater treatment technologies toward greater sustainability and resilience.