Currently, an antimicrobial effectiveness that indicated by the diameter of the inhibition zones of antimicrobial was measured using manual tools such as rulers or calipers. In this paper, a camera-based system for virtual measurement of the diameter of the inhibition zones is introduced using a non-metric camera. The hardware consists of a set of measuring instruments, light source, and a camera that connected to PC directly. The software consists of image processing techniques performing camera calibration, spatial calibration, virtual ruler interface and transforming pixel value to a meter value. A standard checkerboard with certain dimension and shape was used as calibrator object to generate camera and spatial calibration parameter. The measurement system was evaluated using a set of standard checkerboard object at various camera object distances, camera angles and object orientation. The average measurement error between actual and computed inhibition diameter obtained from the antimicrobial object are 0.932%, 0.847% and 1.136% at camera-object distance 15 cm, 20 cm and 25 cm, respectively. Finally, the proposed measurement system is very promising and could be used as an instrument for antimicrobial effectiveness evaluation.