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Urolithiasis, referred to as the formation of stones in the urinary tract, is a common disease with growing prevalence and high recurrence rate worldwide. Although researchers have endeavoured to explore the mechanism of urinary stone formation for novel effective therapeutic and preventative measures, the exact aetiology and pathogenesis remain unclear. Propelled by sequencing technologies and culturomics, great advances have been made in understanding the pivotal contribution of the human microbiome to urolithiasis. Indeed, there are diverse and abundant microbes interacting with the host in the urinary tract, overturning the dogma that urinary system, and urine are sterile. The urinary microbiome of stone formers was clearly distinct from healthy individuals. Besides, dysbiosis of the intestinal microbiome appears to be involved in stone formation through the gut-kidney axis. Thus, the human microbiome has potential significant implications for the aetiology of urolithiasis, providing a novel insight into diagnostic, therapeutic, and prognostic strategies. Herein, we review and summarize the landmark microbiome studies in urolithiasis and identify therapeutic implications, challenges, and future perspectives in this rapidly evolving field. To conclude, a new front has opened with the evidence for a microbial role in stone formation, offering potential applications in the prevention, and treatment of urolithiasis.