Agricultural spraying involves two major tasks: guiding a sprayer in response to a GPS navigation device, and simultaneous monitoring of rear-attached booms under various illumination and terrain difficulty levels. The aim of the present study was to investigate the effect of illumination, task difficulty, and task level on the mental workload of an individual operating an agricultural sprayer in response to a commercial GPS lightbar, and to explore the sensitivity of the NASA-TLX and SSWAT subjective rating scales in discriminating the subjective experienced workload under various task, illumination, and difficulty levels. Mental workload was measured using performance measures (lateral root mean square error and reaction time), physiological measures (0.1 Hz power of HRV, latency of the P300 component of event-related potential, and eye-glance behavior), and two subjective rating scales (NASA-TLX and SSWAT). Sixteen male university students participated in this experiment, and a fixed-base high-fidelity agricultural tractor simulator was used to create a simulated spraying task. All performance measures, the P300 latency, and subjective rating scales showed a common trend that mental workload increased with the change in illumination from day to night, with task difficulty from low to high, and with task type from single to dual. The 0.1 Hz power of HRV contradicted the performance measures. Eye-glance data showed that under night illumination, participants spent more time looking at the lightbar for guidance information. A similar trend was observed with the change in task type from single to dual. Both subjective rating scales showed a common trend of increasing mental workload with the change in illumination, difficulty, and task levels. However, the SSWAT scale was more sensitive than the NASA-TLX scale. With the change in illumination, difficulty, and task levels, participants spent more mental resources to meet the increased task demand; hence, the illumination, task difficulty, and task level affected the mental workload of an agricultural sprayer operator operating a sprayer in response to a GPS lightbar.