Quercetin, a ubiquitous flavonoid, is known to have antibacterial effects. The purpose of this study was to investigate the effect of quercetin on cecal microbiota of Arbor Acre (AA) broiler chickens in vivo and the bacteriostatic effect and antibacterial mechanism of quercetin in vitro. In vivo, 480 AA broilers (1 day old) were randomly allotted to four treatments (negative control and 0.2, 0.4, or 0.6 g of quercetin per kg of diet) for 42 days. Cecal microbial population and distribution were measured at the end of the experiment. The cecal microflora in these broilers included Proteobacteria, Fimicutes, Bacteroidetes, and Deferribacteres. Compared with the negative control, quercetin significantly decreased the copies of Pseudomonas aeruginosa (P < 0.05), Salmonella enterica serotype Typhimurium (P < 0.01), Staphylococcus aureus (P < 0.01), and Escherichia coli (P < 0.01) but significantly increased the copies of Lactobacillus (P < 0.01), Bifidobacterium (P < 0.01), and total bacteria (P < 0.01). In vitro, we investigated the bacteriostatic effect of quercetin on four kinds of bacteria (E. coli, P. aeruginosa, S. enterica Typhimurium, and S. aureus) and the antibacterial mechanism of quercetin in E. coli and S. aureus. The bacteriostatic effect of quercetin was stronger on gram-positive bacteria than on gram-negative bacteria. Quercetin damaged the cell walls and membranes of E. coli (at 50 × MIC) and S. aureus (at 10 × MIC). Compared with the control, the activity of the extracellular alkaline phosphatase and β-galactosidase and concentrations of soluble protein in E. coli and S. aureus were significantly increased (all P < 0.01), and the activity of ATP in S. aureus was significantly increased (P < 0.01); however, no significant change in ATP activity in E. coli was observed (P > 0.05). These results suggest that quercetin has potential as an alternative antibiotic feed additive in animal production.