Monophasic tin dioxide nanoflowers (TONFs) assembled by rod-like nanostructures were prepared by coprecipitation method using tin chloride and ammonia precipitators, as the starting materials, without using any surfactants or templates. The structural, compositional, optical, and morphological properties of TONFs were investigated by XRD, FT-IR, UV-vis, SEM-EDX, and TEM techniques. Synthesized TONFs demonstrated inhibition of quorum sensing- (QS-) regulated virulence in pathogens, viz., Chromobacterium violaceum, Pseudomonas aeruginosa, and Serratia marcescens. Significant reduction in biofilm formation in all test pathogens was also observed which was further validated by CLSM images illustrating disturbed biofilm architecture. Vital functions like EPS, swarming motility, and cell surface hydrophobicity that contribute to successful biofilm formation were reduced after addition of sub-MICs of TONFs. Significant eradication of preformed biofilms was recorded upon addition of subinhibitory concentrations of TONFs in all test pathogens. The study highlights the broad-spectrum QS and biofilm inhibition by TONFs that can be exploited in future to protect food from contamination and combatting persistent drug-resistant infections.