Abstract: Bacterial colony counting is one of the fundamental approaches utilized in microbiology for quantifying and isolating various sets of microorganisms. Until now, in most laboratories, the process of counting is achieved manually and this process is very exhausted and consumes time. In this study, an accurate technique based on digital image analysis is proposed for auto-counting of the bacterial colony with minimal time and effort. The results show better reliability by containing the obtained measurements of counting comparing with the manual counting with a total average error equal to 1.22%. The proposed technique outperforms the other traditional techniques, giving a hopeful technique for several related applications.