The nematode Caenorhabditis elegans is a useful model host for pathogenesis research that can be infected by a large number of human pathogens. Conventionally, nematode–pathogen infection assays are mainly performed on agar medium which are labor-intensive and time-consuming. To overcome these challenges, we develop for the first time an integrated microfluidic device for evaluating in vivo antimicrobial activity of natural compounds, which allows infection and anti-infection assays to be sequentially and automatically carried out in liquid medium. The device consists of a worm dispenser with 32 trap-construction chambers and concentration gradient generators, in which the processes of introduction, dispensation, confinement of worms in the chamber and drug delivery to the chamber can be integrated into a single device. In addition, the operation of the device is simple and does not require any expensive robotic fluid handling systems to dispense samples. To demonstrate the ability of this device, we devise an on-line screening experiment using a Caenorhabditis elegans–Staphylococcus aureus infection model and characterize the survival rate of the infected worms treated with antibiotics. Then, we applied the system to evaluate the antibacterial activity of several components of rhubarb: aloe-emodin, rhein and emodin at various concentrations. The device is able to load uniform worms into each chamber within 10 min and then generate various chemical concentrations automatically and simultaneously. Furthermore, the on-chip method only requires 6 h to establish the infection model and 48 h to perform the subsequent treatments. Based on the excellent advantages and scalable properties of microfluidics, the microfluidic platform holds a great potential in high-throughput screening for antimicrobials.