Autophagy is an evolutionarily conserved pathway to degrade damaged proteins and organelles for subsequent recycling in cells during times of nutrient deprivation. This process plays an important role in tumor development and progression, allowing cancer cells to survive in nutrient-poor environments. The plant kingdom provides a powerful source for new drug development to treat cancer. Several plant extracts induce autophagy in cancer cells. However, little is known about the role of plant extracts in autophagy inhibition, particularly autophagy-related (ATG) proteins. In this study, we employed S-tagged gamma-aminobutyric acid receptor associated protein like 2 (GABARAPL2) as a reporter to screen 48 plant extracts for their effects on the activity of autophagy protease ATG4B. Xanthium strumarium and Tribulus terrestris fruit extracts were validated as potential ATG4B inhibitors by another reporter substrate MAP1LC3B-PLA2. The inhibitory effects of the extracts on cellular ATG4B and autophagic flux were further confirmed. Moreover, the plant extracts significantly reduced colorectal cancer cell viability and sensitized cancer cells to starvation conditions. The fruit extract of X. strumarium consistently diminished cancer cell migration and invasion. Taken together, the results showed that the fruit of X. strumarium may have an active ingredient to inhibit ATG4B and suppress the proliferation and metastatic characteristics of colorectal cancer cells.