This paper presents a novel colony analysis system including an adjustable image acquisition subsystem and a wavelet-watershed-based image segmentation algorithm. An illumination box was constructed-both front lightning and back lightning illuminations can be chosen by users based on the properties of Petri dishes. In the illumination box, the lightning is uniform, which makes image processing easy. A digital camera at the top of the box is connected to a PC computer; all the camera functions are controlled by the developed computer software in this study. As usual, in the image processing part, the hardest task is image segmentation which is carried out by the four different algorithms: 1. recursive image segmentation on gray similarity; 2. canny edge detection-based segmentation; 3. the combination of 1 and 2, and 4. colony delineation on wavelet and watershed. The first three algorithms can obtain good results for ordinary colony images, and for the images including a lot of small (tiny) colonies and dark colonies and overlapping (or touching) colonies, the algorithm 4 can obtain better results than the others. The algorithms are tested by using a large number of different colony images, and the testing results are satisfactory.