The human fibroblast collagenase catalytic domain (MMP1ca) that is considered a prototype for all interstitial collagenase and plays an important role in the turnover of collagen fibrils in the matrix was expressed as an inclusion body in the Escherichia coli. The purified enzyme displayed activity with substrate Dnp-Pro-Leu-Ala-Leu-Trp-Ala-Arg-OH with a Km value of 26.61 ± 1.42 μM. The inhibition activity of the nine flavonoid compounds and gallic acid against MMP1ca was examined. Among the compounds tested, the IC50 of seven flavonoid compounds were determined and ranged from 14.13 to 339.21 μM. Epigallocatechin gallate (EGCG) showed the highest inhibition toward MMP1ca with IC50 values of 14.13 ± 0.49 μM. EGCG showed a competitive inhibition pattern with a Ki value of 10.47 ± 0.51 μM. The free binding energy of EGCG against MMP1ca was −13.07 kcal mol−1, which was calculated by using Autodock 3.0.5 software and showed numerous hydrophobic and hydrogen bond interactions. The galloyl group of EGCG, gallocatechin gallate and epicatechin gallate was determined to be important for inhibitory activity against MMP1ca.