This study attempted to multimodally measure mental workload and validate indicators for estimating mental workload. A simulated computer work composed of mental arithmetic tasks with different levels of difficulty was designed and used in the experiment to measure physiological signals (heart rate, heart rate variability, electromyography, electrodermal activity, and respiration), subjective ratings of mental workload (the NASA Task Load Index) and task performance. The indices from electrodermal activity and respiration had a significant increment as task difficulty increased. There were no significant differences between the average heartbeats and the low-frequency/high-frequency ratio among tasks. The classification of mental workload using combined indices as inputs showed that classification models combining physiological signals and task performance can reach satisfying accuracy at 96.4% and an accuracy of 78.3% when just taking physiological indices as inputs. The present study also shows that ECG and EDA signals have good discriminating power for mental workload detection. The methods used in this study could be applied to office workers and the findings provide preliminary support and theoretical exploration for follow-up early mental workload detection systems, whose implementation in the real world could beneficially impact worker health and company efficiency.