Bacterial colony classification is crucial in microbiology but remains labor-intensive and time-consuming when performed manually. Deep learning, particularly Convolutional Neural Networks (CNNs), enables automated classification, improving accuracy and efficiency. This study compares MobileNetV2 and EfficientNet-B0 for bacterial colony classification, evaluating the impact of data augmentation on model performance. Using the Neurosys AGAR dataset, preprocessing techniques such as histogram equalization, gamma correction, and Gaussian blur were applied, while data augmentation (rotation, noise addition, luminosity adjustments) improved model generalization. The dataset was split (80% training, 20% testing), and models were trained with learning rates (0.0001, 0.001) and epochs (100, 150, 200). Results show EfficientNet-B0 outperforms MobileNetV2, achieving higher validation accuracy and stability, with optimal performance at 150–200 epochs and a lower learning rate (0.0001). Data augmentation significantly improved accuracy and reduced overfitting. While MobileNetV2 remains a lightweight alternative, its performance is heavily reliant on augmentation. These findings highlight EfficientNet-B0 as the superior model, supporting the automation of microbiological diagnostics. Future research should explore hybrid CNN architectures, Vision Transformers (ViTs), and real-time implementation for improved classification efficiency.