Broccoli extract mainly contains polyphenols and glucosinolates (GSLs). GSLs can be hydrolyzed by gut microorganisms into isothiocyanates (ITCs) and other active substances. These substances have anticancer, anti-inflammatory, antimicrobial, and atherosclerosis-reducing functions. In this study, a high concentration (2000 μmol/L GSLs and 24 μmol/L polyphenols) and a low concentration (83 μmol/L GSLs and 1 μmol/L polyphenols) of broccoli extract were prepared. Gut microorganisms from fresh human feces were cultured to simulate the gut environment in vitro. The GSL content decreased and the types and content of ITCs increased with broccoli extract hydrolysis through cyclic condensation and gas chromatography-mass spectrometry (GC-MS) analyses. Broccoli extract significantly increased probiotics and inhibited harmful bacteria through 16S rDNA sequencing. Based on phylum level analysis, Firmicutes and Lachnospiraceae increased significantly (P < 0.05). At the genus level, both high- and low-concentration groups significantly inhibited Escherichia and increased Bilophila and Alistipes (P < 0.05). The high-concentration group significantly increased Bifidobacterium (P < 0.05). The broccoli extract improved the richness of gut microorganisms and regulated their structure. The GSL hydrolysis was significantly correlated with Bilophila, Lachnospiraceae, Alistipes, Bifidobacterium, Escherichia, and Streptococcus (P < 0.05). These study findings provide a theoretical foundation for further exploring a probiotic mechanism of broccoli extract in the intestine.