Macrolactins have attracted considerable attention due to their value and application in medicine and agriculture. However, poor yields severely hinder their broader application in these fields. This study aimed to improve macrolactins production in Bacillus siamensis using a combined atmospheric and room-temperature plasma mutagenesis and a microbial microdroplet culture system. After 25 days of treatment, a desirable strain with macrolactins production 3.0-fold higher than that of the parental strain was successfully selected. The addition of 30mg/L ZnSO