Identification and analysis of biological microscopy images need high focus and years of experience to master the art. The rise of deep neural networks enables analyst to achieve the desired results with reduced time and cost. Light sheet fluorescence microscopies are one of the types of 3D microcopy images. Processing microscopy images is tedious process as it consists of low-level features. It is necessary to use proper image processing techniques to extract the low-level features of the biological microscopy images. Deep neural networks (DNN) are efficient in extracting the features of images and able to classify with high accuracy. Convolutional neural networks (CNN) are one of the types of neural networks that can provide promising results with less error rates. The ability of CNN to extract the low-level features of images makes it popular for image classification. In this paper, a CNN-based 3D bacterial image classification is proposed. 3D images contain more in-depth features than 2D images. The proposed CNN model is trained on 3D light sheet fluorescence microscopy images of larval zebrafish. The proposed CNN model classifies the bacterial and non-bacterial images effectively. Intense experimental analyses are carried out to find the optimal complexity and to get better classification accuracy. The proposed model provides better results than human comprehension and other traditional machine learning approaches like random forest, support vector classifier, etc. The details of network architecture, regularization, and hyperparameter optimization techniques are also presented. © Springer Nature Singapore Pte Ltd 2021.