Herbicides have been widely used in Eucalyptus plantations, but little is known about the effects of herbicides on understory plant and soil microbial communities in Eucalyptus plantations. This study compared and analyzed the effects of herbicide application at different dosages and frequencies on understory plant and soil microbial communities through application experiments with low-dose high-frequency (LHF), medium-dose medium-frequency (MMF), and high-dose low-frequency (HLF) herbicides and comparison with manual tending (MT) in Eucalyptus plantations. The results showed that herbicide application resulted in significant changes in understory plant species and functional group composition in Eucalyptus plantations. However, herbicides did not significantly reduce the species richness or diversity of understory plant communities, but decreasing the herbicide application frequency and increasing the recovery time led the species richness and diversity index to show a trend of recovery. Herbicide application also results in a decrease in soil nutrient contents. With the increase of herbicide concentration and the decrease of herbicide frequency, the soil pH and total potassium concentration showed a trend of increasing, the soil water content, available phosphorus and available potassium concentrations showed a trend of decreasing, but the concentrations of soil organic matter, total nitrogen and available nitrogen showed a trend of decreasing first and then increasing. Herbicides indirectly affected soil microbial communities through their negative effects on understory plant communities and soil nutrients. LHF significantly reduced the importance value of the vine functional group while significantly increased that of the fern functional group, thus significantly reduced the phospholipid fatty acid (PLFA) content of the microbial community, fungi, and actinomycetes. MMF significantly decreased the importance value of the woody and vine functional groups but significantly increased the Gramineae plant functional group, leading to a significant decrease in the PLFA content of the microbial community and actinomycetes. HLF did not significantly affect the understory plant or soil microbial communities but significantly reduced both the soil total phosphorus concentration and the soil available phosphorus content. Meanwhile, herbicide significantly reduced the soil microbial biomass carbon and nitrogen content. Therefore, the application of herbicides should be decreased to reduce the negative effects on understory plant and soil microbial communities.