Urolithiasis, the formation of urinary calculi, remains a prevalent and clinically significant urological disorder worldwide. Despite extensive research, the etiology of urolithiasis remains multifactorial and not entirely understood. Emerging evidence suggests that the urinary microbiome, previously considered sterile, plays a pivotal role in the pathogenesis and progression of urolithiasis. This article delves into the intricate relationship between urolithiasis and the urinary microbiome, elucidating the key microbial players, their potential mechanisms of action, and the clinical implications of this association. We explore recent advancements in metagenomics, metabolomics, and microbial profiling techniques that have revolutionized our understanding of the urinary microbiome in urolithiasis. A comprehensive understanding of the interplay between urolithiasis and the urinary microbiome holds promise for novel diagnostic and therapeutic strategies, offering new avenues for the prevention and management of this debilitating condition.