Microbial genomics and functional genetics have undergone transformative advances over the past decade, driven by breakthroughs in genome sequencing technologies, multi-omics profiling, and gene-function validation tools. This review synthesizes recent progress (2022–2025) in microbial genome sequencing, functional genomics, transcriptomics, proteomics, metabolomics, and gene-function discovery, highlighting their integration to unravel microbial biology at unprecedented resolution. We discuss how high-throughput sequencing has expanded the microbial genomic landscape, enabling the characterization of uncultured taxa via metagenomics and single-cell genomics. Furthermore, we explore how multi-omics approaches (transcriptomics-proteomics-metabolomics) have facilitated the annotation of novel genes, elucidation of metabolic pathways, and identification of genotype-phenotype relationships in diverse microbial systems—from extremophiles to host-associated microbiomes. We also address technical challenges and emerging solutions in gene-function validation, such as CRISPR-based tools and synthetic biology approaches, and their applications in ecological research and biotechnology. Finally, we outline future directions for microbial genomics, including the integration of artificial intelligence with multi-omics data to accelerate gene discovery and predict microbial behavior. This review underscores the pivotal role of microbial genomics and functional genetics in advancing our understanding of microbial diversity, ecology, and evolution, while unlocking new opportunities for biotechnological innovation.