Microbial proteomics is a key approach to understanding and analysing microbial physiology, metabolism, pathogenic mechanisms, and environmental adaptation. Due to limitations in sample purity, throughput, and cost, traditional structural analysis techniques struggle to fully detect the vast number of unknown proteins within the microbial proteome. In recent years, structural elucidation of the microbial proteome has been driven by innovations in cryo-electron microscopy, high-resolution mass spectrometry, AI-based structure prediction, and multi-model integration techniques. This field has evolved from single-protein studies to the entire proteome, from in vitro purification to in situ dynamics, and from static structures to functional networks. The following article provides a systematic review of the latest advances in cryo-EM visualisation of proteomics, in situ mass spectrometry, AI-based structure prediction, and multi-technique integration, offering a reference for the in-depth decoding of microbial protein functional networks.