Pseudomonas syringae (P. syringae) can infect over 50 different crops worldwide, causing significant economic losses, and it is one of the major bacterial pathogens affecting global crops. Currently, there are no effective control strategies for bacterial leaf spot disease caused by P. syringae. In this study, a high-throughput screening of 1680 natural products was conducted to evaluate their antibacterial effects. Microbial-derived compounds such as Polymyxin B sulfate (1), Methacycline hydrochloride (2), Tetracycline hydrochloride (3), Chlortetracycline hydrochloride (4), Demeclocycline hydrochloride (5), and Doxycycline hyclate (6) exhibited strong antibacterial activity against Pseudomonas syringae pv. tomato DC3000 (Pst DC3000), with inhibition rates exceeding 60%. Further research revealed that compound 4 had a minimum inhibitory concentration of 0.4 μM. After 12 h of treatment with 12.8 μM of compound 4, bacterial killing efficiency reached 98%. Moreover, compound 4 effectively inhibited the occurrence of bacterial leaf spot disease. In conclusion, this study suggests that microbial-derived natural products hold great potential as effective bactericides against Pst DC3000, providing a new approach for the control of bacterial leaf spot disease.